Mammalian sphingosine kinase type 2 isoforms, cloning, expression and methods of use thereof

ABSTRACT

Nucleic acids encoding mouse and human sphingosine kinase type 2 isoforms, methods for detecting agents or drugs which inhibit or promote sphingosine activity and therapeutic agents containing peptides or antibodies to peptides encoded by such nucleic acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application of application Ser. No.10/830,677 filed Apr. 22, 2004, which is a Divisional application ofapplication Serial No. 09/817,676 filed Mar. 26, 2001 (U.S. Pat. No.6,800,470), which claims the benefit under 35 USC 119(e) of ProvisionalApplication Ser. No. 60/194,318 filed Apr. 3, 2000, said benefit under35 U.S.C. 119(e) is also claimed herein.

GOVERNMENT RIGHTS

This invention was made with United States government support underGrant GM 43880 from the National Institutes of Health and a PostdoctoralFellowship BC961968 from the United States Army Medical Research andMateriel Command, Prostate Cancer Research Program (VEN). The UnitedStates government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention concerns mammalian (such as mouse and human)sphingosine kinase type 2 isoforms, the molecular cloning of suchisoforms and methods of use of such isoforms. Sphingosine kinase type 2has distinct characteristics when compared to sphingosine kinase type 1.

2. Background Information

Sphingosine-1-phosphate (SPP) is a bioactive sphingolipid metabolitewhich regulates diverse biological processes acting both inside cells asa second messenger to regulate proliferation and survival and outsidecells as a ligand for G-protein coupled receptors of the EDG-1 subfamily(Spiegel, S., J. Leukoc. Biol., 65, (1999), 341-344; Goetzl, E. J., An,S. FASEB J., 12, (1998), 1589-1598). Thus, SPP plays important roles asa second messenger to regulate cell growth and survival (Olivera, A.,Spiegel, S., Nature, 365, (1993), 557-560; Cuvillier, O., Pirianov, G.,Kleuser, B., Vanek, P. G., Coso, O. A., Gutkind, S., and Spiegel, S.,Nature, 381, (1996), 800-803).

Many external stimuli, particularly growth and survival factors,activate sphingosine kinase (“SPHK”), the enzyme that forms SPP fromsphingosine. This rapidly growing list includes platelet-derived growthfactor (“PDGF”) (Olivera, A., Spiegel, S., Nature, 365, (1993), 557-560;Pyne, S., Chapman, J. Steele, L., and Pyne, N. J., Eur. J. Biochem.,237, (1996), 819-826; Coroneos, E., Martinez, M., McKenna, S. andKester, M., J. Biol. Chem., 270, (1995), 23305-23309), nerve growthfactor (“NGF”) (Edsall, L. C., Pirianov, G. G., and Spiegel, S., J.Neurosci., 17, (1997), 6952-6960; Rius, R. A., Edsall, L. C., andSpiegel, S., FEBS Lett., 417, (1997), 173-176), vitamin D3 (Kleuser, B.,Cuvillier, O., and Spiegel, S., Cancer Res., 58, (1998) 1817-1824),muscarinic acetylcholine agonists (Meyer zu Heringdorf, D., Lass, H.,Alemany, R., Laser, K. T., Neumann, E. Zhang, C., Schmidt, M., Rauen,U., Jakobs, K. H., and van Koppen, C. J., EMBO J., 17, (1998),2830-2837), TNF-a (Xia, P., Gamble, J. R., Rye, K. A., Wang, L., Hii, C.S. T., Cockerill, P., Khew-Goodall, Y., Bert, A. G., Barter, P. J., andVadas, M. A., Proc. Natl. Acad. Sci. USA, 95, (1998), 14196-14201), andthe cross-linking of the immunoglobulin receptors FceR1 (Choi, O. H.,Kim, J.-H., and Kinet, J.-P., Nature, 380, (1996), 634-636) and FcgR1(Melendez, A., Floto, R. A., Gillooly, D. J., Harnett, M. M., and Allen,J. M., J. Biol. Chem., 273 (1998), 9393-9402).

Intracellular SPP, in turn, mobilizes calcium from internal storesindependently of InsP3 (Meyer zu Heringdorf, D., Lass, H., Alemany, R.,Laser, K. T., Neumann, E. Zhang, C., Schmidt, M., Rauen, U., Jakobs, K.H., and van Koppen, C. J., EMBO J., 17, (1998), 2830-2837; Mattie, M.,Brooker, G, and Spiegel, S., Biol. Chem., 269, (1994), 3181-3188), aswell as eliciting diverse signaling pathways leading to proliferation(Rani, C. S., Berger, A., Wu, J., Sturgill, T. W., Beitner-Johnson, D.,LeRoith, D., Varticovski, L., and Spiegel, S., J. Biol. Chem., 272,(1997), 10777-10783; Van Brocklyn, J. R., Lee, M. J., Menzeleev, R,Olivera, A., Edsall, L., Cuvillier, O., Thomas, D. M., Coopman, P. J.P., Thangada, S., Hla, T., and Spiegel, S., J. Cell Biol., 142, (1998),229-240) and suppression of apoptosis (Cuvillier, O., Pirianov, G.,Kleuser, B., Vanek, P. G., Coso, O. A., Gutkind, S., and Spiegel, S.,Nature, 381, (1996), 800-803; Edsall, L. C., Pirianov, G. G., andSpiegel, S, J. Neurosci., 17, (1997), 6952-6960; Van Brocklyn, J. R.,Lee, M. J., Menzeleev, R., Olivera, A., Edsall, L., Cuvillier, O.,Thomas, D. M., Coopman, P. J. P., Thangada, S., Hla, T., and Spiegel S.,J. Cell Biol., 142, (1998), 229-240).

Moreover, competitive inhibitors of sphingosine kinase block formationof SPP and selectively inhibit calcium mobilization, cellularproliferation and survival induced by these various stimuli (Spiegel,S., J. Leukoc. Biol., 65, (1999), 341-344). Thus, it has been suggestedthat the dynamic balance between levels of the sphingolipid metabolites,ceramide and SPP, and the consequent regulation of opposing signalingpathways, is an important factor that determines the fate of cells(Cuvillier, O., Rosenthal, D. S., Smulson, M. E., and Spiegel, S., J.Biol. Chem., 273, (1998), 2910-2916). For example, stress stimuliincrease ceramide levels leading to apoptosis, whereas survival factorsstimulate SPHK leading to increased SPP levels, which suppress apoptosis(Cuvillier, O., Rosenthal, D. S., Smulson, M. E., and Spiegel, S., J.Biol. Chem., 273, (1998), 2910-2916).

Furthermore, the SPHK pathway, through the generation of SPP, iscritically involved in mediating TNF-α-induced endothelial cellactivation (Xia, P., Gamble, J. R., Rye, K. A., Wang, L., Hii, C. S. T.,Cockerill, P., Khew-Goodall, Y., Bert, A. G., Barter, P. J., and Vadas,M. A., Proc. Natl. Acad. Sci. USA, 95, (1998), 14196-14201) and theability of high density lipoproteins (HDL) to inhibit cytokine-inducedadhesion molecule expression has been correlated with its ability toreset this sphingolipid rheostat (Xia, P., Gamble, J. R., Rye, K. A.,Wang, L., Hii, C. S. T., Cockerill, P., Khew-Goodall, Y., Bert, A. G.,Barter, P. J., and Vadas, M. A., Proc. Natl. Acad. Sci. USA, 95, (1998),14196-14201). This has important implications for the protectivefunction of HDL against the development of atherosclerosis andassociated coronary heart disease. Recent data has also connected thesphingolipid rheostat to allergic responses (Prieschl, E., E., Csonga,R., Novotny, V., Kikuchi, G. E., and Baumruker, T., J. Exp. Med., 190,(1999), 1-8).

Interest in SPP has accelerated recently with the discovery that it is aligand of the G-protein coupled cell surface receptor EDG-1 (VanBrocklyn, J. R., Lee, M. J., Menzeleev, R., Olivera, A., Edsall, L.,Cuvillier, O., Thomas, D. M., Coopman, P. J. P., Thangada, S, Hla, T.,and Spiegel, S., J. Cell Biol., 142, (1998), 229-240; Lee, M. J., VanBrocklyn, J. R., Thangada, S., Liu, C. H., Hand, A. R., Menzeleev, R.,Spiegel, S., and Hla, T., Science, 279, (1998), 1552-1555). This rapidlyled to the identification of several other related receptors, namedEDG-3, -5, -6, and -8, which are also specific SPP receptors (Goetzl, E.J., and An, S., FASEB J., 12, (1998), 1589-1598; Spiegel, S., andMilstein, S., Biochem. Biophys. Acta., 1484(2-3):107-16, (2000)).Sphinganine-1-phosphate, which is structurally similar to SPP and lacksonly the trans double bond at the 4-position, but not lysophosphatidicacid or sphingosylphosphorylcholine, also binds to these receptors (VanBrocklyn, J. R., Tu, Z., Edsall, L. C., Schmidt, R. R., and Spiegel, S.,J. Biol. Chem., 274, (1999) 4626-4632), demonstrating that EDG-1 belongsto a family of G-protein coupled receptors that bind SPP with highaffinity and specificity (Goetzl, E. J. and An, S., FASEB J., 12,(1998), 1589-1598; Spiegel, S. and Milstien, S., Biochem. Biophys.Acta., 1484(2-3):107-116, (2000)).

The EDG-1 family of receptors are differentially expressed, mainly inthe cardiovascular and nervous systems, and are coupled to a variety ofG-proteins and thus can regulate diverse signal transduction pathwaysculminating in pleiotropic responses depending on the cell type andrelative expression of EDG receptors. Although the biological functionsof the EDG-1 family of GPCRs are not completely understood, recentstudies suggest that binding of SPP to EDG-1 stimulates migration andchemotaxis (Wang, F., Van Brocklyn, J. R., Hobson, J. P., Movafagh, S.,Zukowska-Grojec, Z., Milstien, S., and Spiegel, S. J. Biol. Chem., 274,(1999), 35343-35350; English, D., Kovala, A. T., Welch, Z., Harvey, K.A., Siddiqui, R. A., Brindley, D. N., and Garcia, J. G., J. Hematother.Stem Cell Res., 8, (1999), 627-634), and as a consequence, may regulateangiogenesis (Wang. F., Van Brocklyn, J. R., Hobson, J. P., Movafagh,S., Zukowska-Grojec, Z., Milstien, S., and Spiegel, S. J. Biol. Chem.,274, (1999), 35343-35350; Lee, O. H., Kim, Y. M., Lee, Y. M., Moon, E.J., Lee, D. J., Kim, J. H., Kim, K. W., and Kwon, Y. G., Biochem.Biophys. Res. Commun., 264, (1999) 743-750; Lee, M. J., Thangada, S.,Claffey, K. P., Ancellini, N., Liu, C. H., Kluk, M., Volpi, Sha'afi, R.I., and Hla, T., Cell, 99, (1999), 301-312). EDG-5 may play a role incytoskeletal reorganization during neurite retraction, which isimportant for neuronal differentiation and development (Van Brocklyn, J.R., Tu, Z., Edsall, L. C., Schmidt, R. R., and Spiegel, S., J. Biol.Chem., 274, (1999), 4626-4632; MacLennan, A. J., Marks, L., Gaskin, A.A., and Lee, N., Neuroscience, 79, (1997), 217-224).

Critical evaluation of the role of SPP requires cloning of the enzymesthat regulate its metabolism. Recently, rat kidney SPHK has beenpurified to apparent homogeneity (Olivera, A., Kohama, T., Tu, Z.,Milstien, S., and Spiegel, S., J. Biol. Chem., 273, (1998), 12576-12583)and subsequently the first mammalian SPHK, designated mSPHK1 (Kohama,T., Olivera, A., Edsall, L., Nagiec, M. M., Dickson, R., and Spiegel,S., J. Biol. Chem., 273, (1998), 23722-23728) was cloned. Independently,two genes, termed LCB4 and LCB5, were also shown to code for SPHKs inSaccharomyces cerevisiae (Nagiec, M. M., Skrzypek, M., Nagiec, E. E.,Lester, R. L., and Dickson, R. C., J. Biol. Chem., 273, (1998)19437-19442). Moreover, databases identify homologues of mSPHK1 innumerous widely disparate organisms, including worms, plants andmammals, demonstrating that the enzyme is encoded by a member of ahighly conserved gene family (Kohama, T., Olivera, A., Edsall, L.,Nagiec, M. M., Dickson, R., and Spiegel, S., J. Biol. Chem., 273,(1998), 23722-23728). Comparison of the predicted amino acid sequencesof the known SPHK1s revealed five blocks of highly conserved amino acids(Kohama, T., Olivera, A., Edsall, L., Nagiec, M. M., Dickson, R., andSpiegel, S., J. Biol. Chem., 273, (1998), 23722-23728). However, severallines of evidence indicate that there may be multiple mammalian SPHKisoforms.

The finding that SPHK activity in platelets could be chromatographicallyfractionated into several forms with differing responses to detergentsand inhibition by known SPHK inhibitors, indicate the presence ofmultiple enzyme forms in human platelets (Banno, Y., Kato, M., Hara, A.,and Nozawa, Y., Biochem. J., 335, (1998), 301-304). Moreover, homologysearches against a comprehensive nonredundant database revealed thatseveral of the expressed sequence tags (dbEST) at NCBI had significanthomology to conserved domains of mSPHK1a (Kohama, T., Olivera, A.,Edsall, L., Nagiec, M. M., Dickson, R., and Spiegel, S., J. Biol. Chem.,273, (1998), 23722-23728), yet had substantial sequence differences.

U.S. Pat. No. 5,374,616 concerns compositions containingsphingosylphosphorylcholine for promoting cellular proliferation ofmammalian cells.

WO 99/61581 describes DNA fragments which encoded murine sphingosineSPHK1a (381 amino acids) and SPHK1b (388 amino acids).

SUMMARY OF THE INVENTION

It is an object of the present invention to provide isolated andpurified DNAs which encode mammalian (such as a mouse or human)sphingosine kinase type 2 isoforms and peptides encoded therefrom.

It is a further object of the present invention to provide recombinantDNA constructs comprising a vector and the above described DNAs and hostcells transformed with such recombinant DNA constructs.

It is a still further object of the present invention to furnish amethod for producing mouse and human sphingosine type 2 isoform peptidesby culturing such host cells.

It is an additional object of the present invention to provide a methodfor detecting an agent or a drug which inhibits or promotes sphingosinekinase activity.

It is yet another object of the present invention to provide a methodfor regulating a biological process; for treating or ameliorating adisease resulting from increased or decreased cell proliferation orincreased or decreased cell death; and for treating or ameliorating adisease resulting from abnormal migration or motility of cells such ascancer, restenosis or diabetic neuropathy.

The present invention is also directed to an isolated and purified DNAwhich encodes a peptide of a sphingosine kinase type 2 isoform, the DNAcomprising a sequence selected from the group consisting of the sequenceof Genbank Accession No. bankit325787 and the sequence of GenbankAccession No. bankit325752.

The present invention also concerns methods for detecting an agent or adrug which inhibits or promotes sphingosine kinase type 2 activitycomprising:

-   -   (a) providing a recombinant DNA construct as discussed above,        into a cell such that sphingosine kinase type 2 isoform is        produced in the cell;    -   (b) adding at least one drug or agent to the cell, and    -   (c) detecting whether or not the drug or agent inhibits or        promotes sphingosine kinase type 2 activity by measuring        sphingosine kinase-dependent phosphorylation of lipids in the        cells and comparing the resultant measurement to a control which        did not receive the drug or agent, wherein a decrease in the        amount of sphingosine kinase-dependent phosphorylation of lipids        as compared to the control indicates an inhibitory drug or        agent, or an increase in the amount of sphingosine        kinase-dependent phosphorylation of lipids in the cell as        compared to the control indicates a stimulatory drug or agent.

As described hereinabove, the present invention also relates to methodsof regulating a biological process (such as mitogenesis, apoptosis,neuronal development, chemotaxis, angiogenesis and inflammatoryresponses) in a mammal comprising administering to a mammal (such as ahuman) in need thereof, a pharmaceutically effective amount of a peptideas described above.

Also as described hereinabove, the present invention is further directedto methods for the treatment or amelioration of a disease resulting fromincreased cell death or decreased cell proliferation, comprisingadministering to a mammal (such as a human) in need thereof, apharmaceutically effective amount of a peptide as described above.

Further as described above, the present invention also relates tomethods for the treatment or administration of a disease resulting fromdecreased cell death or increased cell proliferation comprisingadministering to a mammal (such as a human) in need thereof, apharmaceutically effective amount of an antibody to a peptide asdescribed above.

Additionally as described above, the present invention further concernsmethods for treatment or amelioration of a disease resulting fromabnormal migration or motility of cells selected from the groupconsisting of cancer, restenosis and diabetic neuropathy, the methodcomprising administering to a mammal (such as a human) in need thereof,a pharmaceutically effective amount of an antibody to a peptide asdescribed above.

The present invention further relates to compositions for (a) regulatingbiological processes, (b) treating or ameliorating diseases resultingfrom increased cell death or decreased cell proliferation, (c) treatingor ameliorating diseases resulting from decreased cell death orincreased cell proliferation, or (d) treating or ameliorating diseasesresulting from abnormal migration or motility of cells (such. as cancer,restenosis and diabetic neuropathy) comprising (i) a pharmaceuticallyeffective amount of a peptide as described above or an antibody to suchpeptide as described above, and (ii) a pharmaceutically acceptablecarrier.

The present invention also involves a method for screening agents ordrugs which reduce or eliminate sphingosine kinase type 2 activity, themethod comprising detecting a decrease in sphingosine kinase type 2enzyme activity in the presence of the agent or drug.

Furthermore, the present invention is directed to a method for detectingthe presence of sphingosine kinase type 2 isoform in a samplecomprising:

-   -   (i) contacting a sample with antibodies which recognize        sphingosine kinase type 2; and    -   (ii) detecting the presence or absence of a complex formed        between sphingosine kinase type 2 and antibodies specific        therefor.

The present invention also concerns a method for detecting sphingosinekinase type 2 in a sample comprising subjecting the sample to apolymerase chain reaction and detecting for the presence of sphingosinekinase type 2.

The present invention is additionally directed to a diagnostic kit fordetecting sphingosine kinase type 2 RNA/cDNA in a sample comprisingprimers or oligonucleotides specific for sphingosine kinase type 2 RNAor cDNA suitable for hybridization to sphingosine kinase type 2 RNA orcDNA and/or amplification of sphingosine kinase type 2 sequences andsuitable ancillary reagents.

Sphingosine kinase catalyzes the phosphorylation of sphingosine to yieldSPP. Based on sequence homology to murine and human sphingosine kinase-1(SPHK1), which was recently cloned (Kohama, et al., J. Biol. Chem., 273,23722-23728, (1998)), the present invention is directed to the cloning,functional characterization, and tissue distribution of a second type ofmouse and human sphingosine kinase (mSPHK2 and hSPHK2).

mSPHK2 and hSPHK2 of the present invention encode proteins of 617 and618 amino acids, respectively, both much larger than SPHK1, and bothcontain the conserved domains previously found in SPHK1, but theirsequences diverge considerably in the centers and at the amino termini.Northern blot analysis of multiple human and murine tissues revealedthat SPHK2 mRNA expression was strikingly different from that of SPHK1and was highest in brain, heart, kidney, testes, and liver. WhereasSPHK1 expression is greatest at mouse embryonic day 7, SPHK2 expressionis only detectable at embryonic day 11 and increases thereafter.

Human embryonic 293 kidney cells transiently transfected with mSPHK2 orhSPHK2 expression vectors had marked increases in SPHK activityresulting in elevated SPP levels. Notably, SPHK2 had somewhat differentsubstrate specificity than SPHK1. D-erythro-sphingosine(dihydrosphingosine, DHS) was an even better substrate thanD-erythro-sphingosine for SPHK2, while DHS was a potent inhibitor ofSPHK1.

SPHK2 also catalyzed the phosphorylation of phytosphingosine and D,L-threo-dihydrosphingosine, albeit to a lesser extent. DMS, acompetitive inhibitor of SPHK1, surprisingly was a non-competitiveinhibitor of SPHK2. Although increasing ionic strength inhibited SPHK1,KCl and NaCl markedly stimulated SPHK2 activity. Moreover, Triton X-100and BSA inhibited SPHK2, in contrast to their effects on SPHK1, whereasphosphatidylserine stimulated both types. The data herein indicate thatSPHK2 is a novel member of this growing class of lipid kinases, which isimportant in the regulation of diverse biological processes, includingmitogenesis, apoptosis, neuronal development, chemotaxis, angiogenesis,and inflammatory responses.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purposes of illustrating the invention, features, aspects andadvantages are shown in the drawings. It is to be understood, however,that the present invention is not limited to that which is depicted inthe drawings

FIG. 1A shows predicted amino acid sequences of murine and human type 2sphingosine kinase based on non-ClustalW alignment of the predictedamino acid sequences of (“mSPHK2”) (SEQ ID NO: 12) and human sphingosinekinase 2 (“hSPHK2”) (SEQ ID NO: 14). Identical and conserved amino acidsubstitutions are shaded dark and light gray, respectively. The dashesrepresent gaps in sequences and numbers on the right refer to the aminoacid sequence of mSPHK2 (SEQ ID NO: 12). The conserved domains (C1 toC5) are indicated by lines. FIG. 1A also shows the amino acid sequenceof mSPHK1 (SEQ ID NO: 15).

FIG. 1B is a schematic representation of conserved regions of SPHK1 andSPHK2. The primary sequence of mSPHK2 is compared to that of mSPHK1a.

FIGS. 2A, 2B and 2C are Northern blots which show the tissue specificexpression of type 1 and type 2 sphingosine kinase.

In FIG. 2A, mSPHK2 (upper panel) and mSPHK1a (middle panel) probes wereend labeled and hybridized to poly(A)+ RNA blots from the indicatedmouse tissues as described hereinbelow. Lanes: 1, heart; 2, brain; 3,spleen; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; 8, testis. Aβ-actin probe (lower panel) was used as a loading control.

FIG. 2B shows the tissue specific expression of hSPHK2. Lanes 1, brain;2, heart; 3, skeletal muscle; 4, colon; 5, thymus; 6, spleen; 7, kidney;8, liver; 9, small intestine; 10, placenta; 11, lung; 12, leukocyte.

FIG. 2C shows the expression of mSPHK1a and mSPHK2 during mouseembryonic development. Poly(A)+ RNA blots from days 7, 11, 15 and 17mouse embryos were probed as in FIG. 2A.

FIGS. 3A and 3B are graphs which show the enzymatic activity ofrecombinant SPHK2.

In FIG. 3A, HEK 293 cells were transiently transfected with an emptyvector or with mSPHK2 or hSPHK2 expression vectors. After 24 hours, SPHKactivity was measured in cytosol (open bars) and particulate fractions(filled bars). The data are means±S.D. Parental and vector-transfectedcells had basal SPHK activities of 26 and 37 pmol/min/mg, respectively.

FIG. 3B shows the changes in mass levels of SPP after transfection withSPHK2. Mass levels of SPP in HEK293 cells transfected with an emptyvector (open bars) or with mSPHK2 (filled bars) or with hSPHK2 (hatchedbars) were measured as described hereinbelow. The data are expressed aspmol/nmol phospholipid.

FIGS. 4A to 4D are graphs which show the substrate specificity ofmSPHK2.

FIG. 4A is a graph which shows SPHK-dependent phosphorylation of varioussphingosine analogs or other lipids (50 mM) which was measured incytosol from HEK293 cells transfected with mSPHK2. Lanes: 1,D-erythro-sphingosine (“D-erythro-Sph”); 2, D-erythro-dihydrosphingosine(“D-erythro-DHS”); 3, D, L-threo-DHS; 4, N,N-dimethylsphingosine(“DMS”); 5, C2-ceramide; 6, C16-ceramide; 7, diacylglycerol; 8,phosphatidylinositol; 9, phytosphingosine. Data are expressed aspercentage of phosphorylation of D-erythro-Sph.

FIGS. 4A to 4D are graphs which show the noncompetitive inhibition ofrecombinant SPHK2 by N,N-dimethylsphingosine.

FIG. 4B shows the dose-dependent inhibition of mSPHK2 by DMS. SPHKactivity in HEK293 cell lysates after transfection as in FIG. 4A wasmeasured with 10 μM D-erythro-sphingosine in the presence of increasingconcentrations of DMS.

FIG. 4C shows a kinetic analysis of DMS inhibition. SPHK activity wasmeasured with varying concentrations of D-erythro-sphingosine in theabsence (open circles) or presence of 10 μM (filled squares) or 20 μMDMS (filled triangles).

FIG. 4D are Lineweaver-Burk plots. The Km for D-erythro-sphingosine was3.4 μM. The Ki value for DMS was 12 μM.

FIGS. 5A to 5E are graphs which show the pH dependence and salt effectson mSPHK2.

FIG. 5A shows cytosolic SPHK2 activity in transfected HEK 293 cells thatwas measured in a kinase buffer with the pH adjusted using the followingbuffers: 200 mM sodium acetate (pH 4.5-5.5, open circles); 200 mM MES(pH 6-7, filled circles); 200 mM potassium phosphate (pH 6.5-8, opensquares); 200 mM HEPES (pH 7-7.5, filled squares); 200 mM Tris-HCl (pH7.5-9, open triangles); and 200 mM borate (pH 10, filled triangle).

FIGS. 5B to 5E show that salts stimulate SPHK2, but inhibit SPHK1.

In FIGS. 5B and 5C, the SPHK activity in HEK293 cell lysates wasmeasured 24 hours after transfection with mSPHK1 (FIG. 5B) or mSPHK2(FIG. 5C) in the absence or presence of increasing concentrations ofNaCl (open squares) or KCl (filled circles).

FIG. 5D shows a kinetic analysis of SPHK2 activation by KCl. mSPHK2activity was measured with varying concentrations ofD-erythro-sphingosine in the absence (open circles), or presence of 50mM KCl (open squares), or 200 mM KCl (filled squares).

FIG. 5E are Lineweaver-Burk plots of data from FIG. 5D. The Km value notaffected by the presence of KCl. Vmax values were 0.1, 0.3 and 1(nmol/min/mg) in the presence of 0, 50, and 200 mM KCl, respectively.

FIGS. 6A to 6B are graphs which show that Triton X-100 and bovine serumalbumin (“BSA”) have differential effects on the activity of SPHK1 andSPHK2. HEK293 cells were transfected with mSPHK1a (open circles) ormSPHK2 (filled circles) and the activities of each in cell lysates weremeasured after 24 hours in the presence of the indicated concentrationsof Triton X-100 (FIG. 6A) or BSA (FIG. 6B).

FIG. 6C is a graph which shows that phosphatidylserine has similareffects on the activity of SPHK1 and SPHK2. HEK293 cells weretransfected with mSPHK1a (circles) or mSPHK2 (triangles) and theactivities of each in cell lysates were measured after 24 hours in thepresence of the indicated concentrations of phosphatidylserine (filledsymbols) or phosphatidylcholine (open symbols). Data are expressed aspercentage of control activity measured without any additions.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention relates to a DNA or cDNAsegment which encodes mammalian (such as mouse and human) sphingosinekinase type 2 isoforms.

In addition, isolated nucleic acid molecules of the invention includeDNA molecules which comprise sequences substantially different fromthose described above but which, due to the degeneracy of the geneticcode, still encode mammalian sphingosine kinase type 2 isoforms. Ofcourse, the genetic code and species-specific codon preferences are wellknown in the art. Thus, it would be routine for one of ordinary skill inthe art to generate the degenerate variants described above, forinstance, to optimize codon expression for a particular host (e.g.,change codons in the human mRNA to those preferred by a bacterial hostsuch as E. coli).

Nucleic acid molecules of the present invention may be in the form ofRNA, such as mRNA, or in the form of DNA, including, for instance, cDNAand genomic DNA obtained by cloning or produced synthetically. The DNAmay be double-stranded or single-stranded. Single-stranded DNA or RNAmay be the coding strand, also known as the “sense strand”, or it may bethe noncoding strand, also referred to as the “antisense strand”.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained. in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

The present invention is further directed to nucleic acid moleculesencoding portions or fragments of the nucleotide sequences describedherein. Fragments include portions of the nucleotide sequences of FIG.1A for mSPHK2 and hSPHK2 at least 10 contiguous nucleotides in lengthselected from any two integers, one of which representing a 5′nucleotide position and a second of which representing a 3′ nucleotideposition, where the first nucleotide for each nucleotide sequence inFIG. 1A is position 1. That is, every combination of a 5′ and 3′nucleotide position of a fragment at least 10 contiguous nucleotidebases in length or any integer between 10 and the length of an entirenucleotide sequence of mSPHK2 or hSPHK2 of FIG. 1A minus 1.

Further, the present invention includes polynucleotides comprisingfragments specified by size, in nucleotides, rather than by nucleotidepositions. The present invention includes any fragment size, incontiguous nucleotides, selected from integers between 1 and the entirelength of an entire nucleotide sequence minus 1. Preferred sizes include20 to 50 nucleotides; sizes of 50 to 300 nucleotides are useful asprimers and probes. Regions from which typical sequences may be derivedinclude, but are not limited to, for example, regions encoding specificepitopes or domains within said sequence, such as domains C1-C5 shown inFIG. 1A.

In another aspect, the invention provides isolated nucleic acidmolecules comprising polynucleotides which hybridize under stringenthybridization conditions to a polynucleotide sequence of the presentinvention described above, for instance, a nucleic acid sequence shownin FIG. 1A or a specified fragment thereof. By “stringent hybridizationconditions” is intended overnight incubation at 42° C. in a solutioncomprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 μg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 0.1×SSC at about 65° C.

The sequences encoding the polypeptides of the present invention orportions thereof may be fused to other sequences which provideadditional functions known in the art such as a marker sequence, or asequence encoding a peptide which facilitates purification of the fusedpolypeptide, peptides having antigenic determinants known to providehelper T-cell stimulation, peptides encoding sites forpost-translational modifications, or amino acid sequences in whichtarget the fusion protein to a desired location, e.g., a heterologousleader sequence.

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of the sphingosine kinase type 2 isoform polypeptides shownin FIG. 1A. Variants may occur naturally, such as a natural allelicvariant. By an “allelic variant” is intended one of several alternateforms of a gene occupying a given locus of a chromosome of an organism.Non-naturally occurring variants may be produced by known mutagenesistechniques. Such variants include those produced by nucleotidesubstitution, deletion or addition of one or more nucleotides in thecoding or noncoding regions or both. Alterations in the coding regionsmay produce conservative or nonconservative amino acid substitutions,deletions, or additions. Especially preferred among these are silentsubstitutions, additions, and deletions which do not alter theproperties and activities of sphingosine kinase type 2 isoformpolypeptides disclosed herein or portions thereof. Also preferred inthis regard are conservative substitutions.

Nucleic acid molecules with at least 90-99% identity to a nucleic acidmolecule which encodes a sphingosine kinase type 2 isoform shown in FIG.1A is another aspect of the present invention. These nucleic acids areincluded irrespective of whether they encode a polypeptide havingsphingosine kinase activity. By “a polypeptide having sphingosine kinasetype 2 activity” is intended polypeptides exhibiting activity similar,but not identical, to an activity of the sphingosine kinase type 2isoform of the present invention, as measured in the assays describedbelow. The biological activity or function of the polypeptides of thepresent invention are expected to be similar or identical to,polypeptides from other organisms that share a high degree of structuralidentity/similarity.

In another embodiment, the present invention relates to a recombinantDNA molecule that includes a vector and a DNA sequence as describedabove. The vector can take the form of a plasmid, phage, cosmid, YAC, aneukaryotic expression vector such as a DNA vector, Pichia pastoris, or avirus vector such as for example, baculovirus vectors, retroviralvectors or adenoviral vectors, and others known in the art. The clonedgene may optionally be placed under the control of (i.e., operablylinked to) certain control sequences such as promoter sequences, orsequences which may be inducible and/or cell type-specific. Suitablepromoters are known to a person with ordinary skill in the art. Theexpression construct will further contain sites for transcriptioninitiation, termination and, in the transcribed region, a ribosomebinding site for translation. Among the vectors preferred for useinclude pCMV-SPORT2 (Life Technologies, Inc.), pcDNA3 (Invitrogen), toname a few.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, electroporation, infection, and othermethods known in the art and described in standard laboratory manualssuch as Current Protocols in Molecular Biology, Ausubel, F. M. et al.(Eds), Wiley & Sons, Inc. All documents cited herein supra and infra arehereby incorporated in their entirety by reference thereto.

In a further embodiment, the present invention relates to host cellsstably transformed or transfected with the above-described recombinantDNA constructs. The host cell can be prokaryotic (for example,bacterial), lower eukaryotic (for example, yeast or insect) or highereukaryotic (for example, all mammals, including, but not limited to, ratand human).

Both prokaryotic and eukaryotic host cells may be used for expression ofdesired coding sequences, when appropriate control sequences which arecompatible with the designated host are used. Among prokaryotic hosts,E. coli is most frequently used. Expression control sequences forprokaryotes include promoters, optionally containing operator portions,and ribosome binding sites. Transfer vectors compatible with prokaryotichosts are commonly derived from, for example, pBR322, a plasmidcontaining operons conferring ampicillin and tetracycline resistance,and the various pUC vectors, which also contain sequences conferringantibiotic resistance markers. These markers may be used to obtainsuccessful transformants by selection. See, for example, Maniatis,Fitsch and Sambrook, Molecular Cloning: A Laboratory Manual, (1982) orDNA Cloning, Volumes I and II (D. N. Glover ed., 1985) for generalcloning methods.

A transformant having a plasmid in which a cDNA encoding human SPHK2 isinserted, namely E. coli pCR3.1-hSPHK2 SANK 70200 has been depositedwith the National Institute of Bioscience and Human-Technology, Agencyof Industrial Science and Technology, 1-3, Higashi 1-chome, Tsukuba-shi,Ibaraki-ken 305-8566, Japan, accession number FERM BP-7110, depositedMar. 29, 2000.

The DNA sequence can be present in the vector operably linked to asequence encoding an IgG molecule, an adjuvant, a carrier, or an agentfor aid in purification of SPHK, such as glutathione S-transferase, or aseries of histidine residues also known as a histidine tag. Therecombinant molecule can be suitable for transfecting eukaryotic cells,for example, mammalian cells and yeast cells in culture systems.Saccharomyces cerevisiae, Saccharomyces carlsbergensis, and Pichiapastoris are the most commonly used yeast hosts, and are convenientfungal hosts. Control sequences for yeast vectors are known in the art.Mammalian cell lines are available as hosts for expression are known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC), such as HEK293 cells, and NIH3T3 cells, to name a few. Suitable promoters are also known in the artand include viral promoters such as that from SV40, Rous sarcoma virus(“RSV”), adenovirus (“ADV”), bovine papilloma virus (“BPV”), andcytomegalovirus (“CMV”).

Mammalian cells may also require terminator sequences and poly Aaddition sequences; enhancer sequences which increase expression mayalso be included, and sequences which cause amplification of the genemay also be desirable. These sequences are known in the art.

The transformed or transfected host cells can be used as a source of DNAsequences described above. When the recombinant molecule takes the formof an expression system, the transformed or transfected cells can beused as a source of the protein described below. In another embodiment,the present invention relates to the employment of nucleotide sequencescorresponding to GenBank/EMBL Data Bank accession nos. bankit325787 andbankit325752.

A polypeptide or amino acid sequence expressed from the nucleotidesequences discussed above, refers to polypeptide having an amino acidsequence identical to that of a polypeptide encoded from the sequence,or a portion thereof wherein the portion contains at least 2 to 5 aminoacids, and more preferably at least 8 to 10 amino acids, and even morepreferably at least 15 amino acids, or which is immunologicallyidentifiable with a polypeptide encoded in the sequence.

A recombinant or derived polypeptide is not necessarily translated froma designated nucleic acid sequence; it may be generated in any manner,including, for example, chemical synthesis, or expression of arecombinant expression system. In addition the polypeptide can be fusedto other proteins or polypeptides which increase its antigenicity, suchas adjuvants, for example.

As noted above, the methods of the present invention are suitable forproduction of any polypeptide of any length, via insertion of theabove-described nucleic acid molecules or vectors into a host cell andexpression of the nucleotide sequence encoding the polypeptide ofinterest by the host cell. Introduction of the nucleic acid molecules orvectors into a host cell to produce a transformed host cell can beeffected by calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection or other methods. Such methods are described inmany standard laboratory manuals, such as in Davis et al., Basic MethodsIn Molecular Biology, (1986).

Once transformed host cells have been obtained, the cells may becultivated under any physiologically compatible conditions of pH andtemperature, in any suitable nutrient medium containing assimilablesources of carbon, nitrogen and essential minerals that support hostcell growth. Recombinant polypeptide-producing cultivation conditionsvary according to the type of vector used to transform the host cells.For example, certain expression vectors comprise regulatory regionswhich require cell growth at certain temperatures, or addition ofcertain chemicals or inducing agents to the cell growth medium, toinitiate the gene expression resulting in the production of therecombinant polypeptide. Thus, the term “recombinantpolypeptide-producing conditions,” as used herein, is not meant to belimited to any one set of cultivation conditions. Appropriate culturemedia and conditions for the above-described host cells and vectors arewell-known in the art. Following its production in the host cells, thepolypeptide of interest may be isolated by several techniques. Toliberate the polypeptide of interest from the host cells, the cells arelysed or ruptured. This lysis may be accomplished by contacting thecells with a hypotonic solution, by treatment with a cellwall-disrupting enzyme such as lysozyme, by sonication, by treatmentwith high pressure, or by a combination of the above methods. Othermethods of bacterial cell disruption and lysis that are known to one ofordinary skill may also be used.

Following disruption, the polypeptide may be separated from the cellulardebris by any technique suitable for separation of particles in complexmixtures. The polypeptide may then be purified by well-known isolationtechniques. Suitable techniques for purification include, but are notlimited to, ammonium sulfate or ethanol precipitation, acid extraction,electrophoresis, immunoadsorption, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, immunoaffinity chromatography,size exclusion chromatography, liquid chromatography (LC), highperformance LC (HPLC), fast performance LC (FPLC), hydroxylapatitechromatography and lectin chromatography.

The recombinant or fusion protein can be used as a diagnostic tool andin a method for producing sphingosine-l-phosphate, detectably labeledand unlabeled, and in a method for measuring levels of SPP in samples asdescribed below. In addition, the recombinant protein can be used as atherapeutic agent to reduce cell death and/or increase cellproliferation. The transformed host cells can be used to analyze theeffectiveness of drugs and agents which inhibit SPHK2 function, such ashost proteins or chemically derived agents or other proteins which mayinteract with the cell to down-regulate or alter the expression ofSPHK2, or its cofactors.

In another embodiment, the present invention relates to monoclonal orpolyclonal antibodies specific for the above-described recombinantproteins (or polypeptides). For instance, an antibody can be raisedagainst a peptide described above, or against, a portion thereof of atleast 10 amino acids, preferably 11 to 15 amino acids. Persons withordinary skill in the art using standard methodology can raisemonoclonal and polyclonal antibodies to the protein (or polypeptide) ofthe present invention, or a unique portion thereof. Material and methodsfor producing antibodies are well known in the art (see, for example,Goding in Monoclonal Antibodies: Principles and Practice, Chapter 4,1986).

The level of expression of sphingosine kinase type 2 can be detected atseveral levels. Using standard methodology well known in the art, assaysfor the detection and quantitation of sphingosine kinase type 2 RNA canbe designed and include northern hybridization assays, in situhybridization assays, and PCR assays, among others. See, for example,Maniatis, Fitsch and Sambrook, Molecular Cloning, A Laboratory Manual,(1982) or DNA Cloning, Volumes I and II (D. N. Glover ed. 1985), orCurrent Protocols in Molecular Biology, Ausubel, F. M. et al., (Eds),Wiley & Sons, Inc. for a general description of methods for nucleic acidhybridization.

Polynucleotide probes for the detection of sphingosine kinase type 2 RNAcan be designed from the sequence available at accession numbersAF068748 and/or AF068749 for the mouse sequence (Kohama, T., et al., J.Biol. Chem., 273:23722-23728). For example, RNA isolated from samplescan be coated onto a surface such as a nitrocellulose membrane andprepared for northern hybridization. In the case of in situhybridization of biopsy samples, for example, the tissue sample can beprepared for hybridization by standard methods known in the art andhybridized with polynucleotide sequences which specifically recognizesphingosine kinase type 2 RNA. The presence of a hybrid formed betweenthe sample RNA and the polynucleotide can be detected by any methodknown in the art such as radiochemistry, or immunochemistry, to name afew.

One of skill in the art may find it desirable to prepare probes that arefairly long and/or encompass regions of the amino acid sequence whichwould have a high degree of redundancy in the corresponding nucleic acidsequences. In other cases, it may be desirable to use two sets of probessimultaneously, each to a different region of the gene. While the exactlength of any probe employed is not critical, typical probe sequencesare no greater than 500 nucleotides, even more typically they are nogreater than 250 nucleotides; they may be no greater than 100nucleotides, and also may be no greater than 75 nucleotides in length.Longer probe sequences may be necessary to encompass uniquepolynucleotide regions with differences sufficient to allow relatedtarget sequences to be distinguished. For this reason, probes arepreferably from about 10 to about 100 nucleotides in length and morepreferably from about 20 to about 50 nucleotides.

The DNA sequence of sphingosine kinase type 2 can be used to designprimers for use in the detection of sphingosine kinase type 2 using thepolymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR).The primers can specifically bind to the sphingosine kinase type 2 cDNAproduced by reverse transcription of sphingosine kinase type 2 RNA, forthe purpose of detecting the presence, absence, or quantifying theamount of sphingosine kinase type. 2 by comparison to a standard. Theprimers can be any length ranging from 7 to 40 nucleotides, preferably10 to 35 nucleotides, most preferably 18 to 25 nucleotides homologous orcomplementary to a region of the sphingosine kinase type 2 sequence.

Reagents and controls necessary for PCR or RT-PCR reactions arewell-known in the art. The amplified products can then be analyzed forthe presence or absence of sphingosine kinase type 2 sequences, forexample, by gel fractionation, by radiochemistry, and immunochemicaltechniques. This method is advantageous, since it requires a smallnumber of cells. Once sphingosine kinase type 2 is detected, adetermination of whether the cell is overexpressing or underexpressingsphingosine kinase type 2 can be made by comparison to the resultsobtained from a normal cell using the same method. Increased sphingosinekinase type 2 RNA levels correlate with increased cell proliferation andreduced cell death.

In another embodiment, the present invention relates to a diagnostic kitfor the detection of sphingosine kinase type 2 RNA in cells. The kitcomprises a package unit having one or more containers of sphingosinekinase type 2 oligonucleotide primers for detection of sphingosinekinase type 2 by PCR or RT-PCR or sphingosine kinase type 2polynucleotides for the detection of sphingosine kinase type 2 RNA incells by in situ hybridization or Northern analysis and, in some kits,including containers of various reagents used for the method desired.The kit may also contain one or more of the following items:polymerization enzymes, buffers, instructions, controls and detectionlabels. Kits may include containers of reagents mixed together insuitable proportions for performing the methods in accordance with theinvention. Reagent containers preferably contain reagents in unitquantities that obviate measuring steps when performing the subjectmethods.

In a further embodiment, the present invention provides a method foridentifying and quantifying the level of sphingosine kinase type 2present in a particular biological sample. Any of a variety of methodswhich are capable of identifying (or quantifying) the level ofsphingosine kinase type 2 in a sample can be used for this purpose.

Diagnostic assays to detect sphingosine kinase type 2 may comprisebiopsy or in situ assay of cells from an organ or tissue sections, aswell as an aspirate of cells from a tumor or normal tissue. In addition,assays may be conducted upon cellular extracts from organs, tissues,cells, urine, or serum or blood or any other body fluid or extract.

When assaying a biopsy, the assay will comprise contacting the sample tobe assayed with a sphingosine kinase type 2 ligand, natural orsynthetic, or an antibody, polyclonal or monoclonal, which recognizessphingosine kinase type 2, or an antiserum capable of detectingsphingosine kinase type 2, and detecting the complex formed betweensphingosine kinase type 2 present in the sample and the sphingosinekinase type 2 ligand or antibody added.

Sphingosine kinase type 2 ligands or substrates include for example,sphingosine, in addition to natural and synthetic classes of ligands andtheir derivatives which can be derived from natural sources such asanimal or plant extracts. Other sphingosine kinase type 2 ligandsinclude calmodulin.

Sphingosine kinase type 2 ligands or anti-sphingosine kinase type 2antibodies, or fragments of ligand and antibodies capable of detectingsphingosine kinase type 2 may be labeled using any of a variety oflabels and methods of labeling for use in diagnosis and prognosis ofdisease associated with increased cell proliferation, such as cancer, orreduced cell death. Examples of types of labels which can be used in thepresent invention include, but are not limited to enzyme labels,radioisotopic labels, nonradioactive isotopic labels, andchemiluminescent labels.

Examples of suitable enzyme labels include malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcoholdehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphateisomerase, peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate hydrogenase, glucoamylase, acetylcholine esterase,etc.

Examples of suitable radioisotopic labels include ³H, ¹¹¹In, ¹²⁵I, ³²P,³⁵S, ¹⁴C, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹Ci, ²¹¹At, ²¹²Pb,⁴⁷Sc, ¹⁰⁹Pd, ¹¹C, ¹⁹F and ¹³¹I.

Examples of suitable non-radioactive isotopic labels include ¹⁵⁷Gd,⁵⁵Mn, ¹⁶²Dy, ⁵²Tr and ⁴⁶Fe.

Examples of suitable fluorescent labels include a ¹⁵²Eu label, afluorescein label, an isothiocyanate I label, a rhodamine label, aphycoerythrin label, a phycocyanin label, an allophycocyanin label and afluorescamine label.

Examples of chemiluminescent labels include a luminal label, anisoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridinium salt label, an oxalate ester label, a luciferinlabel and a luciferase label.

Those of ordinary skill in the art will know of other suitable labelswhich may be employed in accordance with the present invention. Thebinding of these labels to ligands and to antibodies or fragmentsthereof can be accomplished using standard techniques commonly known tothose of ordinary skill in the art. Typical techniques are described byKennedy, J. H., et al., (1976), Clin. Chem. Acta., 70, 1-31, and Schurs,A. H. W. M., et al., (1977), Clin. Chem. Acta., 81, 1-40. Couplingtechniques mentioned in the latter are the glutaraldehyde method, theperiodate method, the dalemide method, and others.

The detection of the antibodies (or fragments of antibodies) of thepresent invention can be improved by the use of carriers. Well-knowncarriers include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have virtually any possiblestructural configuration, so long as the coupled molecule is capable ofbinding to sphingosine kinase type 2. Thus, the support configurationmay be spherical, as in a bead, or cylindrical, as in the inside surfaceof a test tube, or the external surface of a rod. Alternatively, thesurface may be flat such as a sheet or test strip. Those of ordinaryskill in the art will know many other suitable carriers for bindingmonoclonal antibody, or will be able to ascertain the same by the use ofroutine experimentation.

The ligands or antibodies, or fragments of antibodies or ligands ofsphingosine kinase type 2 discussed above may be used to quantitativelyor qualitatively detect the presence of sphingosine kinase type 2. Suchdetection may be accomplished using any of a variety of immunoassaysknown to persons of ordinary skill in the art such as radioimmunoassays,immunometic assays, etc. Using standard methodology well known in theart, a diagnostic assay can be constructed by coating on a surface(i.e., a solid support) for example, a microtitration plate or amembrane (e.g., a nitrocellulose membrane), antibodies specific forsphingosine kinase type 2 or a portion of sphingosine kinase type 2, andcontacting it with a sample from a person suspected of having asphingosine kinase type 2 related disease. The presence of a resultingcomplex formed between sphingosine kinase type 2 in the sample andantibodies specific therefor can be detected by any of the knowndetection methods common in the art, such as fluorescent antibodyspectroscopy or colorimetry. A good description of a radioimmune assaymay be found in Laboratory Techniques and Biochemistry in MolecularBiology by Work, T. S., et al., North Holland Publishing Company, N.Y.(1978), incorporated by reference herein. Sandwich assays are describedby Wide at pages 199-206 of Radioimmune Assay Method, edited by Kirkhamand Hunter, E. & S. Livingstone, Edinburgh, 1970.

The diagnostic methods of this invention are predictive of proliferationand metastatic potentials in patients suffering from cancers includingcarcinomas of the lung, such as small cell carcinoma, large cellcarcinoma, squamous carcinoma, and adenocarcinoma, stomach carcinoma,prostatic adenocarcinoma, ovarian carcinoma such as serouscystadenocarcinoma and mucinous cystadenocarcinoma, ovarian germ celltumors, testicular carcinomas, and germ cell tumors, pancreaticadenocarcinoma, biliary adenocarcinoma, heptacellular carcinoma, renalcell adenocarcinoma, endometrial carcinoma including adenocarcinomas andmixed Mullerian tumors (carcinosarcomas), carcinomas of the endocervix,ectocervix, and vagina such as adenocarcinoma and squamous carcinoma,basal cell carcinoma, melanoma, and skin appendage tumors, esophagealcarcinoma, carcinomas of the nasopharynx and oropharynx includingsquamous carcinoma and adenocarcinomas, salivary gland carcinomas, brainand central nervous system tumors, including tumors of glial, neuronal,and meningeal origin, tumors of peripheral nerve, soft tissue sarcomasand sarcomas of bone and cartilage. Cells of these tumors which expressincreased levels of sphingosine kinase type 2, RNA or sphingosine kinasetype 2 protein, have increased proliferation and decreased cell death.

The protein can be used to identify inhibitors of sphingosine kinasetype 2 activity. Using an enzyme assay, natural and synthetic agents anddrugs can be discovered which result in a reduction or elimination ofsphingosine kinase type 2 enzymatic activity. Knowledge of the mechanismof action of the inhibitor is not necessary as long as a decrease in theactivity of sphingosine kinase type 2 is detected. Inhibitors mayinclude agents or drugs which either bind or sequester the enzyme'ssubstrate(s) or cofactor(s), or inhibit the enzyme itself directly, forexample, by irreversible binding of the agent or drug to the enzyme orindirectly, for example, by introducing an agent which binds thesphingosine kinase type 2 substrate. Agents or drugs related to thepresent invention may result in partial or complete inhibition ofsphingosine kinase type 2 activity.

Inhibitors of sphingosine kinase type 2 includeDL-threo-dihydrosphingosine (DHS) and the more recently discoveredinhibitor N,N-dimethylsphingosine (“DMS”) described in Edsall, L. C. etal., (1998), Biochemistry, 37, 12892-12898. Inhibitors of sphingosinekinase type 2 may be used in the treatment or amelioration of diseasessuch as cancer, atherosclerosis, neurodegenerative disorders, i.e.,stroke and Alzheimer's disease.

Agents which decrease the level of sphingosine kinase type 2 (i.e., in ahuman or an animal) or reduce or inhibit sphingosine kinase type 2activity may be used in the therapy of any disease associated with theelevated levels of sphingosine kinase type 2 or diseases associated withincreased cell proliferation, such as cancer. An increase in the levelof sphingosine kinase type 2 is determined when the level of sphingosinekinase type 2 in a tumor cell is about 2 to 3 times the level ofsphingosine kinase type 2 in the normal cell, up to about 10 to 100times the amount of sphingosine kinase type 2 in a normal cell. Agentswhich decrease sphingosine kinase type 2 RNA include, but are notlimited to, one or more ribozymes capable of digesting sphingosinekinase type 2 RNA, or antisense oligonucleotides capable of hybridizingto sphingosine kinase type 2 RNA, such that the translation ofsphingosine kinase type 2 is inhibited or reduced resulting in adecrease in the level of sphingosine kinase type 2. These antisenseoligonucleotides can be administered as DNA, as DNA entrapped inproteoliposomes containing viral envelope receptor proteins (Kanoda, Y.et al., (1989), Science, 5, 243, 375) or as part of a vector which canbe expressed in the target cell such that the antisense DNA or RNA ismade. Vectors which are expressed in particular cell types are known inthe art, for example, for the mammary gland. See Furth, J. Mammary GlandBiol. Neopl., 2, (1997), 373, for examples of conditional control ofgene expression in the mammary gland.

Alternatively, the DNA can be injected along with a carrier. A carriercan be a protein such as a cytokine, for example, interleukin or apolylysine-glycoprotein carrier. Such carrier proteins and vectors andmethods of using same are known in the art. In addition, the DNA couldbe coated onto tiny gold beads and such beads can be introduced into theskin with, for example, a gene gun (Ulmer, T. B. et al., Science, 259,(1993), 1745).

Alternatively, antibodies, or compounds capable of reducing orinhibiting sphingosine kinase type 2, that is reducing or inhibitingeither the expression, production or activity of sphingosine kinase type2, such as antagonists, can be provided as an isolated and substantiallypurified protein, or as part of an expression vector capable of beingexpressed in the target cell, such that the sphingosine kinase type 2reducing or inhibiting agent is produced. In addition, co-factors suchas various ions, i.e., Ca²⁺ or factors which affect the stability of theenzyme can be administered to modulate the expression and function ofsphingosine kinase type 2. These formulations can be administered bystandard routes. In general, the combinations may be administered by thetopical, transdermal, intraperitoneal, oral, rectal, or parenteral(e.g., intravenous, subcutaneous, or intramuscular) route. In addition,sphingosine kinase type 2 inhibiting compounds may be incorporated intobiodegradable polymers being implanted in the vicinity of where drugdelivery is desired, for example, at the site of a tumor so that thesphingosine kinase type 2 inhibiting compound is slowly releasedsystemically. The biodegradable polymers and their use are described,for example, in detail in Brem et al., J. Neurosurg., 74, (1991),441-446. These compounds are intended to be provided to recipientsubjects in an amount sufficient to effect the inhibition of sphingosinekinase type 2. Similarly, agents which are capable of negativelyaffecting the expression, production, stability or function ofsphingosine kinase type 2, are intended to be provided to recipientsubjects in an amount sufficient to effect the inhibition of sphingosinekinase type 2. An amount is said to be sufficient to “effect” theinhibition or induction of sphingosine kinase type 2 if the dosage,route of administration, etc., of the agent are sufficient to influencesuch a response.

In line with the function of sphingosine kinase type 2 in cellproliferation, agents which stimulate the level of sphingosine kinasetype 2, such as agonists of SPHK2, may be used in the therapy of anydisease associated with a decrease of SPHK2, or a decrease in cellproliferation, wherein SPHK2 is capable of increasing suchproliferation, e.g., developmental retardation.

In providing a patient with agents which modulate the expression orfunction of sphingosine kinase type 2 to a recipient patient, the dosageof administered agent will vary depending upon such factors as thepatient's age, weight, height, sex, general medical condition, previousmedical history, etc. In general, it is desirable to provide therecipient with a dosage of agent which is in the range of from about 1pg/kg to 10 mg/kg (body weight of patient), although a lower or higherdosage may be administered.

A composition is said to be “pharmacologically acceptable” if itsadministration can be tolerated by a recipient patient. Such an agent issaid to be administered in a “therapeutically effective amount”, if theamount administered is physiologically significant. An agent isphysiologically significant if its presence results in a detectablechange in the physiology of a recipient patient. The compounds of thepresent invention can be formulated according to known methods toprepare pharmaceutically useful compositions, whereby these materials,or their functional derivatives, are combined in admixture with apharmaceutically acceptable carrier vehicle. Suitable vehicles and theirformulation, inclusive of other human proteins, e.g., human serumalbumin, are described, for example, in Remington's PharmaceuticalSciences, 16th Ed., Osol, A. ed., Mack Easton, Pa. (1980). In order toform a pharmaceutically acceptable composition suitable for effectiveadministration, such compositions will contain an effective amount ofthe above-described compounds together with a suitable amount of carriervehicle.

Additional pharmaceutical methods may be employed. to control theduration of action. Control release preparations may be achieved throughthe use of polymers to complex or absorb the compounds. The controlleddelivery may be exercised by selecting appropriate macromolecules (forexample, polyesters, polyamino acids, polyvinyl, pyrrolidone,ethylenevinylacetate, methylcellulose, carboxymethylcellulose, orprotamine sulfate) and the concentration of macromolecules as well asthe method of incorporation in order to control release. Anotherpossible method to control the duration of action by controlled releasepreparations is to incorporate the compounds of the present inventioninto particles of a polymeric material, such as polyesters, polyaminoacids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.Alternatively, instead of incorporating these agents into polymericparticles, it is possible to entrap these materials in microcapsulesprepared, for example, interfacial polymerization, for example,hydroxymethylcellulose for gelatin-microcapsules andpoly(methylmethacrylate)-microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(1980).

The present invention also provides kits for use in the diagnostic ortherapeutic methods described above. Kits according to this aspect ofthe invention may comprise one or more containers, such as vials, tubes,ampules, bottles and the like, which may comprise one or more of thecompositions of the invention.

The kits of the present invention may comprise one or more compounds orcompositions of the present invention, and one or more excipients,diluents or adjuvants.

Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the present invention.

The following Materials and Methods were used in the Examples describedbelow.

EXAMPLES

Materials

SPP, sphingosine, and N,N-dimethylsphingosine were from Biomol ResearchLaboratory Inc. (Plymouth Meeting, Pa.). All other lipids were purchasedfrom Avanti Polar Lipids (Birmingham, Ala.). [g-32P]ATP (3000 Ci/mmol)was purchased from Amersham (Arlington Heights, Ill.). Poly-L-lysine andcollagen were obtained from Boehringer Mannheim (Indianapolis, Ind.).Restriction enzymes were obtained from New England Biolabs (Beverly,Mass.). Poly(A)+ RNA blots of multiple mouse adult tissues werepurchased from Clontech (Palo Alto, Calif.). “Lipofectamine PLUS” and“Lipofectamine” were obtained from Life Technologies, Inc.(Gaithersburg, Md.).

Example 1 cDNA Cloning of Murine Sphingosine Kinase Type 2 (mSPHK2)

BLAST searches of the EST database identified a mouse EST clone(GenBank® accession number AA839233) which had significant homology toconserved domains of mSPHK1a (Kohama, T., Olivera, A., Edsall, L.,Nagiec, M. M., Dickson, R. and Spiegel, S., J. Biol. Chem., 273, (1998),23721-23728), yet had substantial sequence differences. Using this EST,a second isoform of SPHK, denoted mSPHK2, was cloned by two differentPCR approaches.

In the first approach, the method PCR cloning from a mouse cDNA library(Stratagene, La Jolla, Calif.) was used. Approximately 1×10⁶ phage wereplated on twenty 150 mm plates, plaques were collected, and plasmidswere isolated using standard procedures (Ausubel, F. M., Brent, R.,Kingston, R. E., Moore, D. D., Smith, J. A., Seidman, J. G., and Struhl,K., Current Protocols in Molecular Biology, Green Publishing Associatesand Wiley-Interscience, New York (1987)). An initial PCR reaction wascarried out with a sequence specific primer (M-3-1,5′-CCTGGGTGCACCTGCGCCTGTATTGG (SEQ ID NO: 1)) and the M13 reverseprimer. The longest PCR products were gel purified and used as thetemplate for a second PCR which contained a sequence specific antisenseprimer (M-3-2, 5′-CCAGTCTTGGGGCAGTGGAGAGCC-3′ (SEQ ID NO:2)) and the T3primer. The final PCR products were subcloned using a “TOPO TA” cloningkit (Invitrogen) and then sequenced. Platinum high fidelity DNApolymerase (Life Technologies) was used for the PCR amplifications withthe following cycling parameters: 30 cycles of 94° C. for 30 seconds,55° C. for 45 seconds, and 70° C. for 2 minutes with a final primerextension at 72° C. for 5 minutes.

In a second approach, 5′RACE PCR was performed with the 5′RACE Systemfor Rapid Amplification of cDNA ends according to the manufacturer'sprotocol (Life Technologies). Poly(A)+ RNA was isolated from Swiss 3T3fibroblasts using a Quick Prep mRNA Purification kit (Pharmacia). Thefirst strand cDNA was synthesized at 42° C. for 50 minutes with 5 mg ofSwiss 3T3 poly(A)+ RNA using a target antisense primer designed from thesequence of AA839233 (m-GSP1, 5′-AGGTAGAGGCTTCTGG (SEQ ID NO:3)) andSuperScript II reverse transcriptase (Life Technologies). Twoconsecutive PCR reactions using this cDNA as a template and LA Taqpolymerase (TaKaRa) were carried out as follows: first PCR, 94° C. for 2minutes followed by 30 cycles of 94° C. for 1 minute, 55° C. for 1minute, 72° C. for 2 minutes, and primer extension at 72° C. for 5minutes with 5′RACE Abridged Anchor Primer,5′-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG (SEQ ID NO:4) and the targetspecific antisense primer m-GSP2, 5′-GCGATGGGTGAAAGCTGAGCTG (SEQ IDNO:5); for the second PCR, the same conditions were employed, exceptthat the annealing temperature was 65° C., with Abridged UniversalAmplification Primer (AUAP), 5′-GGCCACGCGTCGACTAGTAC (SEQ ID NO:6) andm-GSP3, 5′-AGTCTCCAGTCAGCTCTGGACC (SEQ ID NO:7). PCR products werecloned into pCR2.1 and sequenced. The PCR products were subcloned intopCR3.1 and pcDNA 3 expression vectors.

Example 2 cDNA Cloning of Human Sphingosine Kinase-2(hSPHK2)

Poly(A)+ RNA from HEK293 cells was used for a 5′RACE reaction. Targetspecific antisense primers (h-GSP1, 5′-CCCACTCACTCAGGCT (SEQ ID NO:8);h-GSP2, 5′-GAAGGACAGCCCAGCTTCAGAG (SEQ ID NO:9); and h-GSP3,5′-ATTGACCAATAGAAGCAACC (SEQ ID NO:10)) were designed according to thesequence of a human EST clone (accession number AA295570). A firststrand cDNA was synthesized with 5 μg of HEK293 mRNA and h-GSP1. ThiscDNA was used as a template in an initial PCR reaction using 5′RACEAbridged Anchor Primer and h-GSP2. Then, a nested PCR was carried outusing the AUAP primer and h-GSP3. The resulting PCR products were clonedand sequenced as described above.

Example 3 Overexpression and Activity of SPHK2

Human embryonic kidney cells (HEK293, ATCC CRL-1573) and NIH 3T3fibroblasts (ATCC CRL-1658) were cultured as described in Olivera, A.,Kohama, T., Edsall, L. C., Nava, V., Cuvillier, O., Poulton, S., andSpiegel, S., J. Cell Biol., 147, (1999), 545-558. HEK293 cells wereseeded at 6×10⁵ per well in poly-L-lysine coated 6 well plates. After 24hours, cells were transfected with 1 μg of vector alone or with vectorscontaining sphingosine kinase constructs and 6 μl of “LipofectaminePLUS” reagent plus 4 μl of “Lipofectamine” reagent per well. One tothree days after transfection, cells were harvested and lysed byfreeze-thawing as described in Kohama, T., Olivera, A., Edsall, L.,Nagiec, M. M., Dickson, R., and Spiegel, S., J. Biol. Chem., 273,(1998), 23722-23728. In some experiments, cell lysates were fractionatedinto cytosol and membrane fractions by centrifugation at 100,000×g for60 minutes. SPHK activity was determined in the presence of sphingosine,prepared as a complex with 4 mg/ml BSA, and [g-32P]ATP in kinase buffer(Olivera, A. and Spiegel, S. in Methods in Molecular Biology, (Bird, I.M. ed.), (1998), Vol. 105, 233-242, Humana Press, Inc., Totowa, N.J.),containing 200 mM KCl, unless indicated otherwise. 32P-SPP was separatedby TLC and quantified with a phosphoimager as previously described.

Example 4 Lipid Extraction and Measurement of SPP

Cells were washed with PBS and scraped in 1 ml of methanol containing2.5 μl concentrated HCl. Lipids were extracted by adding 2 mlchloroform/1M NaCl (1:1, v/v) and 100 μl 3N NaOH and phases separated.The basic aqueous phase containing SPP, and devoid of sphingosine,ceramide, and the majority of phospholipids, was transferred to asiliconized glass tube. The organic phase was re-extracted with 1 mlmethanol/1M NaCl (1:1, v/v) plus 50 μl 3N NaOH, and the aqueousfractions were combined. Mass measurement on SPP in the aqueous phaseand total phospholipids in the organic phase were measured exactly asdescribed in Edsall, L. C., Pirianov, G. G., and Spiegel, S., J.Neurosci., 17, (1997) 6952-6960; Edsall, L. C., and Spiegel, S., Anal.Biochem., 272, (1999) 80-86).

Example 5 Northern Blotting Analysis

Poly(A)+ RNA blots containing 2 μg of poly(A)+ RNA per lane frommultiple adult mouse and human tissues and mouse embryos were purchasedfrom Clontech. Blots were hybridized with the 1.2 kb PSTI fragment ofmouse EST AA389187 (mSPHK1 probe), the 1.5 kb EcoRI fragment ofpCR3.1-mSPHK2, or the 0.3 kb PvuII fragment of pCR3.1-hSPHK1, aftergel-purification and labeling with [a-32P]dCTP. Hybridization in“ExpressHyb” buffer (Clontech) at 65° C. overnight was carried outaccording to the manufacturer's protocol. Blots were reprobed withb-actin as a loading control (Clontech). Bands were quantified using aMolecular Dynamics Phosphoimager.

Results

Cloning of Type 2 Sphingosine Kinase

Blast searches of the EST data base identified several ESTs thatdisplayed significant homology to the recently cloned mSPHK1a sequence.Specific primers were designed from the sequences of these ESTs and wereused to clone a new type of mouse and human sphingosine kinase (namedmSPHK2 and hSPHK2) by the approaches of PCR cloning from a mouse braincDNA library and 5′-RACE PCR.

ClustalW alignment of the amino acid sequences of mSPHK2 and hSPHK2 isshown in FIG. 1A. The open reading frames of mSPHK2 and hSPHK2 encodepolypeptides of 617 and 618 amino acids, respectively, with 83% identityand 90% similarity. Five highly conserved regions (C1 to C5), identifiedpreviously in SPHK1s (Kohama, T., Olivera, A., Edsall, L., Nagiec, M.M., Dickson, R., Spiegel, S., J. Biol. Chem., 273, (1998) 23722-23728),are also present in both type 2 kinases. Interestingly, the invariantGGKGK positively charged motif in the C1 domain of SPHK1 is modified toGGRGL in SPHK2, suggesting that it may not be part of the ATP bindingsite as previously proposed (Kohama, T., Olivera, A., Edsall, L.,Nagiec, M. M., Dickson, R., Spiegel, S., J. Biol. Chem., 273, (1998)23722-23728). A motif search also revealed that a region beginning justbefore the conserved C1 domains of mSPHK2 and hSPHK2 (amino acid 147 to284) also has homology to the diacylglycerol kinase catalytic site.

Compared to SPHK1, both SPHK2s encode much larger proteins containing236 additional amino acids (FIG. 1B). Moreover, their sequences divergeconsiderably from SPHK1 in the center and at the amino termini. However,after amino acid 140 of mSPHK2, the sequences of type 1 and type 2 SPHKhave a large degree of similarity. These sequences (amino acid 9 to 226for mSPHK1; 141 to 360 for mSPHK2), which encompass domains C1 to C4,have 47% identity and 79% similarity (FIG. 1B). In the C terminalportion of the proteins there are also large homologous regions, whichinclude the C5 domain, from amino acid 227 to 3.81 for mSPHK1 and 480 to617 for mSPHK2, with 43% identity and 78% similarity (FIG. 1B). Thedivergence in these domains suggests that SPHK2 probably did not ariseas a simple gene duplication event.

Tissue Distribution of Sphingosine Kinase Type 2

The tissue distribution of SPHK2 mRNA expression in adult mouse wascompared to that of SPHK1 by Northern blotting (FIG. 2A). In mosttissues, including adult liver, heart, kidney, testis and brain, apredominant 3.1 kb SPHK2 mRNA species was detected, indicatingubiquitous expression. However, the level of expression was markedlyvariable and was highest in adult liver and heart and barely detectablein skeletal muscle and spleen (FIG. 2A). In contrast, the expressionpattern of mSPHK1 was quite different, with the highest mRNA expressionin adult lung, spleen, and liver, although expression in the liver didnot predominate as with mSPHK2. mSPHK1 and mSPHK2 were both temporallyand differentially expressed during embryonic development. mSPHK1 wasexpressed highly at mouse embryonic day 7 (E7) and decreaseddramatically after E11 (FIG. 2B). In contrast, at E7, mSPHK2 expressionwas much lower than mSPHK1, and gradually increased up to E17. ThehSPHK2 2.8 kb mRNA transcript was mainly expressed in adult kidney,liver and brain, with much lower expression in other tissues (FIG. 2C).Interestingly, expression of SPHK2 in human kidney was very high andrelatively much lower in the mouse, while the opposite pattern held forthe liver.

Activity of Recombinant Sphingosine Kinase Type 2

To investigate whether mSPHK2 and hSPHK2 encode bona fide SPHKs, HEK293cells were transiently transfected with expression vectors containingthe corresponding cDNAs. Because previous studies have indicated thatSPHK might be present in cells in both soluble and membrane-associatedforms (Olivera, A., and Spiegel, S., Nature, 365, (1993) 557-560; Banno,Y., Kato, M., Hara, A., and Nozawa, Y., Biochem. J., 335, (1998)301-304; Buehrer, B. M., and Bell, R. M., J. Biol. Chem., 267,3154-3159; Olivera, A. Rosenthal, J., and Spiegel, S., Anal. Biochem.,223, (1994) 306-312; Ghosh, T. K., Bian, J., and Gill, D. L., J. Biol.Chem., 269, (1994), 22628-22635), recombinant SPHK2 activity wasmeasured both in cytosol and in membrane fractions of transfected cells.As previously described in Kohama, T., Olivera, A., Edsall, L., Nagiec,M. M., Dickson, R., and Spiegel, S., J. Biol. Chem., 273, (1998)23722-23728, untreated or vector transfected HEK 293 cells have lowlevels of SPHK activity (FIG. 3A). Twenty four hours after transfectionwith mSPHK2 and hSPHK2, in vitro SPHK activity was increased by 20 and35 fold, respectively, and then decreased thereafter (FIG. 3A). Incontrast, SPHK activity from cells transfected with mSPHK1 was muchhigher, 610-fold more than basal levels 24 hours after transfection andremaining at this level for at least 3 more days (data not shown). As inHEK293 cells, transfection of NIH 3T3 fibroblasts with mSPHK1 resultedin much higher SPHK activity than with mSPHK2. It was previously foundthat, similar to untransfected cells, the majority of SPHK activity incells transfected with mSPHK1 was cytosolic (Kohama, T., Olivera, A.,Edsall, L., Nagiec, M. M., Dickson, R., and Spiegel, S., J. Biol. Chem.,273, (1998) 23722-23728). Similarly, in cells transfected with eithermSPHK2 or hSPHK2, 17% and 26%, respectively, of the SPHK activity wasmembrane-associated (FIG. 3B), although Kyte-Doolittle hydropathy plotsdid not suggest the presence of hydrophobic membrane-spanning domains.

Transfection of HEK 293 cells with mSPHK2 and hSPHK2 also resulted in2.2- and 3.3-fold increases in SPP, the product formed by SPHK,respectively (FIG. 3C), was in agreement with previous studies ofsphingolipid metabolite levels after transfections with mSPHK1a showinga lack of correlation of fold increases in levels and in vitro enzymeactivity (Kohama, T., Olivera, A., Edsall, L., Nagiec, M. M., Dickson,R., and Spiegel, S., J. Biol. Chem., 273, (1998) 23722-23728; Olivera,A., Kohama, T., Edsall, L. C., Nava, V., Cuvillier, O., Poulton, S., andSpiegel, S., J. Cell Biol., 147, (1999), 545-558).

Characteristics of Recombinant mSPHK2

Substrate Specificity

Although SPHK2 is highly homologous to SPHK1, there are substantialsequence differences. Therefore, it was of interest to compare theirenzymatic properties. Typical Michaelis-Menten kinetics were observedfor recombinant SPHK2 (data not shown). The Km for D-erythro-sphingosineas substrate is 3.4 μM, almost identical to the Km previously found forSPHK1 (Olivera, A., Kohama, T., Tu, Z., Milstien, S., and Spiegel, S.,J. Biol. Chem., 273, (1998), 12576-12583). Although the naturallyoccurring D-erythro-sphingosine isomer was the best substrate for SPHK1(Kohama, T., Olivera, A., Edsall, L., Nagiec, M. M., Dickson, R., andSpiegel, S., J. Biol. Chem., 273, (1998) 23722-23728),D-erythro-dihydrosphingosine was a better substrate for SPHK2 thanD-erythro-sphingosine (FIG. 4A). Moreover, althoughD,L-threo-dihydrosphingosine and phytosphingosine were notphosphorylated at all by SPHK1, they were significantly phosphorylatedby SPHK2, albeit much less efficiently than sphingosine. Like SPHK1,other lipids including N,N-dimethylsphingosine (DMS), C2- orC16-ceramide, diacylglycerol, and phosphatidylinositol, were notphosphorylated by SPHK2 (FIG. 6A), suggesting high specificity for thesphingoid base.

DMS and DHS have previously been shown to be a potent competitiveinhibitors of SPHK1 (Edsall, L. C., Van Brocklyn, J. R., Cuvillier, O.,Kleuser, B., and Spiegel, S., Biochemistry, 37, (1998), 12892-12898) andhave been used to block increases in intracellular SPP levels resultingfrom various physiological stimuli (Olivera, A., and Spiegel, S.,Nature, 365, (1993), 557-560; Cuvillier, O., Pirianov, G., Kleuser, B.,Vanek, P. G., Coo, O. A., Gutkind, S., and Spiegel, S., Nature, 381,(1996), 800-803; Edsall, L. C., Pirianov, G. G., and Spiegel, S., J.Neurosci, 17, (1997), 6952-6960; Meyer zu Heringdorf, D., Lass, H.,Alemany, R., Laser, K. T., Neumann, E., Zhang, C., Schmidt, M., Rauen,U., Jakobs, K. H., and van Koppen, C. J., EMBO J., 17, 2830-2837; Choi,O, H., Kim, J.-H., and Kinet, J.-P., Nature, 380, (1996), 634-636;Melendez, A., Floto, R. A., Gillooly, D. J., Harnett, M. M., and Allen,J. M., J. Biol. Chem., 273, 9393-9402; Machwate, M., Rodan, S. B.,Rodan, G. A., and Harada, S. I., Mol. Pharmacol., 54, (1998), 70-77).However, because DHS is a substrate for SPHK2 and the product, dihydroSPP, is as potent as SPP in binding to and activating cell surface SPPEDG-1 family receptors, it cannot be used as a tool to investigate therole of SPHK2. Thus, it was important to characterize the inhibitorypotential of the non-substrate DMS on SPHK2. Surprisingly, it was foundthat although DMS was also a potent inhibitor of SPHK2 (FIG. 4B), itacted in a non-competitive manner (FIG. 4C and FIG. 4D). The Ki for DMSwith SPHK2 was 12 μM, slightly higher than the Ki of 4 μM with SPHK1,making it a useful tool to inhibit both types of SPHK.

mSPHK2 had highest activity in the neutral pH range from 6.5 to 8 withoptimal activity at pH 7.5 (FIG. 5A), a pH dependency similar to that ofSPHK1 (data not shown). The activity decreased markedly at pH valuesbelow and above this range.

Effects of KC1 and NaCl

Most of the SPHK activity in human platelets is membrane-associated andextractable with 1 M NaCl (Banno, Y., Kato, M., Hara, A. and Nozawa, Y.,Biochem. J., 335, (1998), 301-304). Furthermore, the salt extractableSPHK from platelets has different properties than the cytosolic enzyme.It was thus of interest to determine the effect of high saltconcentrations on recombinant SPHK1 and SPHK2. Interestingly, it wasfound that high ionic strength had completely opposite effects on theiractivities. SPHK1 was inhibited markedly inhibited by either NaCl andKCl with each causing 50% inhibition at a concentration of 200 mM (FIG.5B). In contrast, SPHK2 activity was dramatically stimulated byincreasing the salt concentration, with a maximal effect at aconcentration of 400 mM, although KCl was much more effective than NaCl.However, above this concentration, SPHK2 activity decreased sharplyalthough remaining elevated even at 1 M salt (FIG. 5C). Thus, theactivities of SPHK1 and SPHK2 have completely opposite responses tochanges in ionic strength. Kinetic analysis of mSPHK2 in the presenceand absence of high concentrations of salt indicated that the Km forsphingosine was unaltered but the Vmax was increased (FIG. 5D and FIG.5E). The physiological significance of these observations remains to bedetermined but it might be related to different subcellularlocalizations.

Substrate Presentation

Because sphingolipids are highly lipophilic, in in vitro SPHK assays,sphingosine is usually presented in micellar form with Triton X-100 oras a complex with BSA (Olivera, A., Rosenthal, J., and Spiegel, S., J.Cell. Biochem., 60, (1996), 529-537; Olivera, A., Barlow, K. D., andSpiegel, S., Methods Enzymol, 311, (2000), 215-223). Furthermore,detergents such as Triton X-100 have been shown to stimulate theactivity of SPHK in rat brain extracts (Buehrer, B. M., and Bell, R. M.,J. Biol. Chem., 267, (1992), 3154-3159) and the enzyme from rat kidney(Olivera, A., Kohama, T., Tu, Z., Milstien, and Spiegel, S., J. Biol.Chem., 273, (1998), 12576-12583), and it was previously found that thestability of rat kidney SPHK was increased in the presence of certaindetergents (Olivera, A., Kohama, T., Tu, Z., Milstien, and Spiegel, S.,J. Biol. Chem., 273, (1998), 12576-12583). However, when the effect ofincreasing concentrations of Triton X-100 on the activities of SPHK1 andSPHK2 were compared, some unexpected results were found. Concentrationsof detergent below 0.005% had no effect, but at higher concentrations,SPHK2 activity was inhibited and SPHK1 activity was markedly increased(FIG. 6A). At a concentration of Triton X-100 of 0.5%, SPHK1 activitywas increased by more than 4 fold while SPHK2 was almost completelyinhibited.

Interestingly, increasing the BSA concentration from the usual SPHKassay conditions with sphingosine-BSA complex as a substrate, i.e. 0.2mg/ml BSA, caused a concentration-dependent inhibition of SPHK2 activitywithout affecting SPHK1 activity (FIG. 6B). Therefore, when measuringSPHK activity in cell or tissue extracts, the method of substratepreparation, whether in mixed micelles or in BSA complexes, must becarefully optimized because the differential effects of Triton X-100 andBSA on activity could yield different results depending on the relativeexpression of the two types of SPHK.

Effects of Phospholipids

Acidic phospholipids, particularly phosphatidylserine, and phosphatidicacid and phosphatidylinositol, and cardiolipin to a lesser extent,induce a dose-dependent increase in SPHK activity in Swiss 3T3fibroblast lysates, whereas neutral phospholipids had no effect(Olivera, A., Rosenthal, J., and Spiegel, S., J. Cell. Biochem., 60,(1996), 529-537). In agreement, recombinant SPHK1 and SPHK2 werestimulated by phosphatidylserine; the activity of both was maximallyincreased 4-fold at a concentration of 40 μg/ml (FIG. 6C) and inhibitedby higher concentrations in a dose-dependent manner. These effects ofphosphatidylserine appeared to be specific since other phospholipids,including phosphatidylcholine, had no effect on the enzyme activity. Incontrast, the activities of the three major forms of SPHK in humanplatelets are not affected by phosphatidylserine (Banno, Y., Kato, M.,Hara, A, and Nozawa, Y., Biochem. J., 335, (1998), 301-304).

The mechanism by which phosphatidylserine enhances the enzymaticactivity of SPHK is not yet understood. One possibility is thatphosphatidylserine possesses unique membrane-structuring propertieswhich better present the substrate, sphingosine. A second possibility isthat SPHK contains determinants that specifically recognize thestructure of the serine headgroup and that these determinants may onlybecome exposed upon interaction of SPHK with membranes. In this regard,the molecular basis for the remarkable specificity of protein kinase Cfor phosphatidylserine has been the subject of much debate. However,recent data reveal that lipid structure and not membrane structure isthe major determinant in the regulation of protein kinase C byphosphatidylserine (Johnson, J. E., Zimmerman, M. L., Daleke, D. L., andNewton, A. C., Biochemistry, 37, (1998), 12020-12025).

The presence of multiple ESTs in the database with significanthomologies to SPHK1 as well as the identification of several genes in S.cerevisiae encoding different SPHKs (Nagiec, M. M., Skrzypek, M.,Nagiec, E. E., Lester, R. L., and Dickson, R. C., J. Biol. Chem., 273,(1998), 19437-19442) suggests that there may be a large and importantSPHK gene family. Although SPHK2 has a high degree of homology to SPHK1,especially in the previously identified conserved domains identified intype 1 SPHKs (Kohama, T., Olivera, A., Edsall, L., Nagiec, M. M.,Dickson, R., and Spiegel, S., J. Biol. Chem., 273, (1998), 23722-23728),it is much larger (65.2 and 65.6 kDa for SPHK1 and SPHK2, respectivelyversus 42.4 kDa for mSPHK1a) and contains an additional 236 amino acids.Furthermore, its differential tissue expression, temporal developmentalexpression, cellular localization, and kinetic properties in response toincreasing ionic strength and detergents, are completely different fromSPHK1, suggesting that it most likely has a different function andregulates levels of SPP in a different manner than SPHK1 which is knownto play a prominent role in regulating cell growth and survival. Thus,type 2 SPHK is considered to be involved in regulation of some of thenumerous biological responses attributed to SPP, such as angiogenesisand allergic responses.

(SEQ ID NO: 11) Sequence for GenBank ® database 1 EMBC Bank AccessionNo. bankit325787    1 aattcggcac gagggaggac cgagtaaacc gaggcttccagaaccaaaga gaagtcagcc   61 tgaggaaagg gctgggaccc ggagcctctc tggcctttccccgtccctgc tctaacactc  121 tccaggggta aagggaccgg agaatcagag acatgatcggagcttgctgg acgagtcgcg  181 tggtgactct ctggccgcac gccgaccgct tctcggtggctcgcggagga cccggtgggc  241 tgtgtgtcgg agcctccgaa gtagctggaa tcaccgtctttcaacacttg gcctggctct  301 gccatttaaa gttgtgatct tggaggctgg tccaggagctgaccacaagc caagagccta  361 ggagtgcttg ggactgaacc agggtcatgg ccccaccaccactactgcca gtggctgcca  421 gcactccaat cctgcacggc gagtttggtt cctacccggccaacggccca cggtttgccc  481 tcaccctcac aacacaagcc ctacacatac agcgactacgcccaaagcca gaagcccggc  541 cccgagatgg tctagtctct ctggatgagg tctcgggctgtggcaccctg cagagccgta  601 gccccgagga cactgcagcc tacttctgca tctacacctacccacgtggc cgtcgagggg  661 gccggcgcag agctacgcgg accttccggg cggatggggccaccacttat gaggagaatc  721 gtgcagaggc ccagcgctgg gccactgccc tcacgtgtctcctccgagga gtgcctctgt  781 caggggacca ggaaatcacc cctgaattgc tgccccggaagcccaggctg ctcatattgg  841 tcaatccctt tggggggcgg ggcctggcct ggcagcgctgtatggaccac gtggtgccaa  901 tgatctctga agctgggctg tccttcaacc tcatacagacagaacgacag aaccatgccc  961 gtgagctggt gcaggggtta agcctgagtg agtgggaaggcattgtcact gtgtctggag 1021 acgggctgct ttacgaggtg ctgaatgggc tccttgatcggccagactgg gaggatgccg 1081 tgcggatgcc cattggtgtc ctcccctgtg gatcgggcaatgcgctagct ggggcggtga 1141 gccatcatgg cgggtttgag caggttgtcg gtgttgacctgttgctcaac tgctcgcttc 1201 ttctctgccg tggtggcagc catcctctgg acttgctctctgtgacgcta gcctcgggat 1261 cccgctgttt ttccttcctg tcagtggcct ggggattcttgtcagatgtg gacattcaca 1321 gtgagcgctt cagggccctg ggcagcgctc gattcacactgggtgcagtg ctaggcctgg 1381 cctcgttgca tacctaccgt ggacgcctct cctacctccccgctaccaca gaaccagcct 1441 tgcccatccc aggccacagt ctgcctcgag ccaagtcagaactagtcttg gctccagccc 1501 cagcccccgc cgccacccac tcgcctctac atcgatctgtgtctgacctg cccctgcccc 1561 ttccccagcc tgccttggtc tcccctggct cccctgagcccctgcctgac ctgtccctca 1621 atggtggtgg tccagagctg actggagact ggggaggagctggggatgca cctctgtccc 1681 cagacccact gctgccttca tcccccaacg ctctcaaaacagctcagctt tcacccatcg 1741 ctgaagggcc cccagaaatg ccagcatctt cggggttcctgcctcccacc cacagtgccc 1801 cagaagcctc tacctggggc ccagtggacc acctcctccctcccctgggc tctccactgc 1861 cccaagactg ggtgacaata gagggggagt ttgtactcatgttgggcatc ttgacgagcc 1921 acctctgcgc agacctgatg gcagccccac atgcacgctttgatgatggc gttgtgcacc 1981 tgtgttgggt gcggagcggc atctcacggg ctgcacttctacgcattttt ctggccatgg 2041 agcatggaaa ccacttcagc ctgggctgcc cccatctgggctatgctgca gcacgtgcct 2101 tccgccttga accactcacg cctcgtggcc tgctcactgtagatggggag ttagtggagt 2161 atgggccaat acaggcgcag gtgcacccag gtctcgccacgctgctcact gggcctgcag 2221 gtcaaaagcc acaagcctga acgagcctaa aagcatggcgagttggtgga accagcgccc 2281 cataggctaa gatctatcat ttacaggtag aagtggggcccgcactcaga actgtgagga 2341 gggtggagag tggtcctgac cctcagttcc cagaggacctagaggctcga gggtggggcc 2401 tgcctttctt gatgtccaat gatggggcct ggaatgtatgagctagcaag gcttcttcag 2461 cttattgacc agccagggtt tcttcttgcc tactccggtgcctctacttg actggccaat 2521 cagcccttga ggggcaggtt cccccaggtg gtccccagatttgcactaat gttcctcccc 2581 tggccagtta gggatgggat gttctgtgtc ttgtgtgtccctctccctag tctaaaaagc 2641 aattgaaaag gtctatgcaa taaaggttgt tgcttccctctaaaaaaaaa aaaaaaaa (SEQ ID NO: 13) Sequence for GenBank ® database 1EMBC Bank Accession No. bankit325752    1 gccaccatgg ccccgcccccaccgccactg gctgccagca ccccgctcct ccatggcgag   61 tttggctcct acccagcccgaggcccacgc tttgccctca cccttacatc gcaggccctg  121 cacatacagc ggctgcgccccaaacctgaa gccaggcccc ggggtggcct ggtcccgttg  181 gccgaggtct caggctgctgcaccctgcga agccgcagcc cctcagactc agcggcctac  241 ttctgcatct acacctaccctcggggccgg cgcggggccc ggcgcagagc cactcgcacc  301 ttccgggcag atggggccgccacctacgaa gagaaccgtg ccgaggccca gcgctgggcc  361 actgccctca cctgtctgctccgaggactg ccactgcccg gggatgggga gatcacccct  421 gacctgctac ctcggccgccccggttgctt ctattggtca atccctttgg gggtcggggc  481 ctggcctggc agtggtgtaagaaccacgtg cttcccatga tctctgaagc tgggctgtcc  541 ttcaacctca tccagacagaacgacagaac cacgcccggg agctggtcca ggggctgagc  601 ctgagtgagt gggatggcatcgtcacggtc tcgggagacg ggctgctcca tgaggtgctg  661 aacgggctcc tagatcgccctgactgggag gaagctgtga agatgcctgt gggcatcctc  721 ccctgcggct cgggcaacgcgctggccgga gcagtgaacc agcacggggg atttgagcca  781 gccctgggcc tcgacctgttgctcaactgc tcactgttgc tgtgccgggg tggtggccac  841 ccactggacc tgctctccgtgacgctggcc tcgggctccc gctgtttctc cttcctgtct  901 gtggcctggg gcttcgtgtcagatgtggat atccagagcg agcgcttcag ggccttgggc  961 agtgcccgct tcacactgggcacggtgctg ggcctcgcca cactgcacac ctaccgcgga 1021 cgcctctcct acctccccgccactgtggaa cctgcctcgc ccacccctgc ccatagcctg 1081 cctcgtgcca agtcggagctgaccctaacc ccagacccag ccccgcccat ggcccactca 1141 cccctgcatc gttctgtgtctgacctgcct cttcccctgc cccagcctgc cctggcctct 1201 cctggctcgc cagaacccctgcccatcctg tccctcaacg gtgggggccc agagctggct 1261 ggggactggg gtggggctggggatgctccg ctgtccccgg acccactgct gtcttcacct 1321 cctggctctc ccaaggcagctctacactca cccgtctccg aaggggcccc cgtaattccc 1381 ccatcctctg ggctcccacttcccacccct gatgcccggg taggggcctc cgacctgcggc 1441 ccgcccgacc acctgctgcctccgctgggc accccgctgc ccccagactg gtgacgctg 1501 gagggggact ttgtgctcatgttggccatc tcgcccagcc acctaggcgc tgacctggtg 1561 gcagctccgc atgcgcgcttcgacgacggc ctggtgcacc tgtgctgggt gcgtagcggc 1621 atctcgcggg ctgcgctgctgcgccttttc ttggccatgg agcgtggtag ccacttcagc 1681 ctgggctgtc cgcagctgggctacgccgcg gcccgtgcct tccgcctaga gccgctcaca 1741 ccacgcggcg tgctcacagtggacggggag caggtggagt atgggccgct acaggcacag 1801 atgcaccctg gcatcggtacactgctcact gggcctcctg gctgcccggg gcgggagccc 1861 tgaaactaaa caagcttggtacccgccggg ggcggggcct acattccaat ggggcggagc 1921 ttgagctagg gggtgtggcctggctgctag agttgtggtg gcaggggccc tggccccgtc 1981 tcaggattgc gctcgctttcatgggaccag acgtgatgct ggaaggtggg cgtcgtcacg 2041 gttaaagaga aatgggctcgtcccgagggt agtgcctgat caatgagggc ggggcctggc 2101 gtctgatctg gggccgcccttacggggcag ggctcagtcc tgacgcttgc cacctgctcc 2161 tacccggcca ggatggctgagggcggagtc tattttacgc gtcgcccaat gacaggacct 2221 ggaatgtact ggctggggtaggcctcagtg agtcggccgg tcagggcccg cagcctcgcc 2281 ccatccactc cggtgcctccatttagctgg ccaatcagcc caggaggggc aggttccccg 2341 gggccggcgc taggatttgcactaatgttc ctctccccgc SEQ ID NO. 14 Amino acid sequences of human SPHK2MAPPPPPLAASTPLLHGEFGSYPARGPRFALTLTSQALHIQRLRPKPEARPRGGLVPLAEVSGCCTLRSRSPSDSAAYFCIYTYPRGRRGARRRATRTFRADGAATYEENRAEAQRWATALTCLLRGLPLPGDGEITPDLLPRPPRLLLLVNPFGGRGLAWQWCKNHVLPMISEAGLSFNLIQTERQNHARELVQGLSLSEWDGIVTVSGDGLLHEVLNGLLDRPDWEEAVKMPVGILPCGSGNALAGAVNQHGGFEFPLGLDLLLNCSLLLCRGGGHPLDLLSVTLASGSRCFSFLSVAWGFVSDVDIQSERFRALGSARFTLGTVLGLATLHTYRGRLSYLPATVEPASPTPAHSLPRAKSELTLTPDPAPPMAHSPLHRSVSDLPLPLPQPALASPGSPEPLPILSLNGGGPELAGDWGGAGDAPLSPDPLLSSPPGSPKAALHSPVSEGAPVIPPSSGLPLPTPDARVGASTCGPPDHLLPPLGTPLPPDWVTLEGDFVLMLAISPSHLGADLVAAPHARFDDGLVHLCWVRSGISRAALLRLFLAMERGSHFSLGCPQLGYAAARAFRLEPLTPRGVLTVDGEQVEYGPLQAQMHPGIGTLLTGPPGCPGREP SEQ ID NO. 12Amino Acid Sequence of mouse SPHK2MAPPPLLPVAASTPILHGEFGSYPANGPRFALTLTTQALHIQRLRPKPEARPRDGLVSLDEVSGCGTLQSRSPEDTAAYFCIYTYPRGRRGGRRRATRTFRADGATTYEENRAEAQRWATALTCLLRGVPLSGDQEITPELLPRKPRLLILVNPFGGRGLAWQRCMDHVVPMISEAGLSFNLIQTERQNHARELVQGLSLSEWEGIVTVSGDGLLYEVLNGLLDRPDWEDAVRMPIGVLPCGSGNALAGAVSHHGGFEQVVGVDLLLNCSLLLCRGGSHPLDLLSVTLASGSRCFSFLSVAWGFLSDVDIHSERFRALGSARFTLGAVLGLASLHTYRGRLSYLPATTEPALPIPGHSLPRAKSELVLAPAPAPAATHSPLHRSVSDLPLPLPQPALVSPGSPEPLPDLSLNGGGPELTGDWGGAGDAPLSPDPLLPSSPNALKTAQLSPIAEGPPEMPASSGFLPPTHSAPEASTWGPVDHLLPPLGSPLPQDWVTIEGEFVLMLGILTSHLCADLMAAPHARFDDGVVHLCWVRSGISRAALLRIFLAMEHGNHFSLGCPHLGYAAARAFRLEPLTPRGLLTVDGELVEYGPIQAQVHPGLATLLTGPAGQKPQA

It will be appreciated that the instant specification is set forth byway of illustration and not limitation, and that various modificationsand changes may be made without departing from the spirit and scope ofthe present invention.

1. An isolated and purified DNA which encodes a mammalian sphingosinekinase type 2 isoform.
 2. The isolated and purified DNA of claim 1,which encodes a mouse sphingosine kinase type 2 isoform.
 3. The isolatedand purified DNA of claim 1, which encodes a human sphingosine kinasetype 2 isoform.
 4. An isolated and purified DNA selected from the groupconsisting of a) a DNA having a nucleotide sequence consistingessentially of the nucleotide sequence of SEQ ID NO: 11 and b) a DNAencoding protein consisting essentially of the amino acid sequence ofSEQ ID NO:
 12. 5. A protein encoded by the DNA of claim
 4. 6. Anisolated and purified DNA selected from the group consisting of a) a DNAhaving a nucleotide sequence consisting essentially of the nucleotidesequence of SEQ ID NO: 13 and b) a DNA encoding protein consistingessentially of the amino acid sequence of SEQ ID NO:
 14. 7. An isolatedprotein comprising an amino acid sequence of murine SPHK2 consistingessentially of SEQ ID NO:
 12. 8. An isolated and purified DNA whichencodes a protein of a sphingosine kinase type 2 isoform, said DNAcomprising a sequence selected from the group consisting of the sequenceof Genbank Accession No. bankit325787 and the sequence of GenbankAccession No. bankit325752.
 9. A recombinant DNA construct comprising:(a) a vector and (b) the DNA of claim
 2. 10. The recombinant DNAconstruct according to claim 9, wherein said vector is an expressionvector.
 11. The recombinant DNA construct according to claim 9, whereinsaid vector is a prokaryotic vector.
 12. The recombinant DNA constructaccording to claim 9, wherein said vector is a eukaryotic vector.
 13. Ahost cell transformed with the recombinant DNA construct according toclaim
 9. 14. The host cell according to claim 13, wherein said cell isprokaryotic.
 15. The host cell according to claim 13, wherein said cellis eukaryotic.
 16. A recombinant DNA construct comprising: (a) a vectorand (b) the DNA of claim
 2. 17. The recombinant DNA construct accordingto claim 16, wherein said vector is an expression vector.
 18. Therecombinant DNA construct according to claim 16, wherein said vector isa prokaryotic vector.
 19. The recombinant DNA construct according toclaim 16, wherein said vector is a eukaryotic vector.
 20. A host celltransformed with the recombinant DNA construct according to claim 16.21. The host cell according to claim 20, wherein said cell isprokaryotic.
 22. The host cell according to claim 20, wherein said cellis eukaryotic.
 23. A method for producing a mouse sphingosine kinasetype 2 isoform protein which comprises culturing the host cell accordingto claim 13, under conditions such that an isolated mouse sphingosinekinase type 2 isoform DNA is expressed and said mouse sphingosine kinasetype 2 isoform protein is thereby produced.
 24. A method for producing ahuman sphingosine kinase type 2 isoform protein which comprisesculturing the host cell according to claim 20, under conditions suchthat an isolated human sphingosine kinase type 2 isoform DNA isexpressed and said human sphingosine kinase type 2 isoform protein isthereby produced.
 25. A method for detecting an agent or a drug whichinhibits or promotes an enzymatic activity of a sphingosine kinase type2 isoform comprising: (i) providing a recombinant DNA constructcomprising a vector and a DNA into a cell such that a sphingosine kinasetype 2 isoform is produced in said cell, wherein said DNA is selectedfrom the group consisting of (a) a DNA having a nucleotide sequenceconsisting essentially of the nucleotide sequence of SEQ ID NO: 13 and(b) a DNA encoding protein consisting essentially of the amino acidsequence of SEQ ID NO: 14; (ii) adding at least one drug or agent tosaid cell, and (iii) detecting whether or not said drug or agentinhibits or promotes the enzymatic activity of the sphingosine kinasetype 2 isoform by measuring sphingosine kinase type 2-dependentphosphorylation of lipids in said cell and comparing the resultantmeasurement to a control which did not receive said drug or agent,wherein a decrease in the amount of sphingosine kinase type 2-dependentphosphorylation of the lipids as compared to the control indicates aninhibitory drug or agent, or an increase in the amount of sphingosinekinase type 2-dependent phosphorylation of the lipids in said cell ascompared to the control indicates a stimulatory drug or agent.
 26. Amethod of regulating a biological process in a mammal comprisingadministering to a mammal in need thereof a pharmaceutically effectiveamount of the protein according to claim
 6. 27. The method of claim 26,wherein the mammal is a human.
 28. The method of claim 27, wherein thebiological process is selected from the group consisting of mitogenesis,apoptosis, neuronal development, chemotaxis, angiogenesis and aninflammatory response.
 29. The method of claim 27, wherein thebiological process is angiogenesis.
 30. A method for the treatment oramelioration of a disease resulting from increased cell death ordecreased cell proliferation, comprising administering to a mammal inneed thereof a pharmaceutically effective amount of a protein accordingto claim
 6. 31. The method of claim 30, wherein the mammal is a human.32. A method for the treatment or amelioration of a disease resultingfrom decreased cell death or increased cell proliferation comprisingadministering to a mammal in need thereof a pharmaceutically effectiveamount of an antibody to a protein according to claim
 6. 33. The methodof claim 32, wherein the mammal is a human.
 34. A method for thetreatment or amelioration of a disease resulting from abnormal migrationor motility of cells selected from the group consisting of cancer,restenosis and diabetic neuropathy, the method comprising administeringto a mammal in need thereof, a pharmaceutically effective amount of anantibody to a protein according to claim
 6. 35. The method of claim 34,wherein the mammal is a human.
 36. The method of claim 35, wherein thedisease is cancer.
 37. A composition for treating or ameliorating adisease resulting from increased cell death or decreased cellproliferation comprising a pharmaceutically effective amount of aprotein according to claim 6, and a pharmaceutically acceptable carrier.38. A method for the treatment or amelioration of developmentalretardation in a human comprising administering to a human in needthereof a pharmaceutically effective amount of the protein according toclaim
 6. 39. A method for screening agents or drugs which reduce oreliminate sphingosine kinase type 2 activity, the method comprisingdetecting a decrease in sphingosine kinase type 2 enzyme activity in thepresence of said agent or drug.
 40. A method for detecting the presenceof a sphingosine kinase type 2 isoform in a sample comprising (i)contacting a sample with an antibody which recognize sphingosine kinasetype 2; and (ii) detecting the presence or absence of a complex formedbetween sphingosine kinase type 2 and an antibody specific therefor. 41.A method for detecting sphingosine kinase type 2 in a sample comprisingsubjecting the sample to a polymerase chain reaction, a northernhybridization assay or an in situ hybridization assay and detecting forthe presence of sphingosine kinase type
 2. 42. A diagnostic kit fordetecting sphingosine kinase type 2 RNA/cDNA in a sample comprising oneor more containers including primers or nucleotides specific forsphingosine kinase type 2 RNA or cDNA suitable for hybridization tosphingosine kinase type 2 RNA or cDNA and/or amplification ofsphingosine kinase type 2 RNA or cDNA and suitable ancillary reagents.43. A method for detecting an agent or a drug which inhibits or promotesan enzymatic activity of a sphingosine kinase type 2 isoform comprising:(i) contacting a sphingosine kinase type 2 isoform protein and lipids inthe presence of at least one drug or agent, wherein the sphingosinekinase type 2 isoform protein is selected from the group consisting of(a) a protein encoded by DNA having a nucleotide sequence consistingessentially of the nucleotide sequence of SEQ ID NO: 13 and (b) aprotein consisting essentially of the amino acid sequence of SEQ ID NO:14; (ii) detecting whether or not said drug or agent inhibits orpromotes the enzymatic activity of the sphingosine kinase type 2 isoformby measuring sphingosine kinase-dependent phosphorylation of the lipidsand comparing the resultant measurement to a control which did notreceive the drug or agent, wherein a decrease in the amount ofsphingosine kinase type 2-dependent phosphorylation of the lipids ascompared to the control indicates an inhibitory drug or agent, or anincrease in the amount of sphingosine kinase type 2-dependentphosphorylation of the lipids as compared to the control indicates astimulatory drug or agent.
 44. A recombinant DNA construct comprising:(a) a vector and (b) the DNA of claim
 4. 45. A recombinant DNA constructcomprising: (a) a vector and (b) the DNA of claim
 5. 46. An antibodyspecific for a protein according to claim
 6. 47. The antibody accordingto claim 46, wherein the antibody is a polyclonal antibody.
 48. Theantibody according to claim 46, wherein the antibody is a monoclonalantibody.
 49. A method of regulating a biological process in a mammalcomprising administering to a mammal in need thereof a pharmaceuticallyeffective amount of the protein according to claim
 7. 50. The method ofclaim 49, wherein the mammal is a human.
 51. A method for the treatmentor amelioration of a disease resulting from increased cell death ordecreased cell proliferation, comprising administering to a mammal inneed thereof a pharmaceutically effective amount of a protein accordingto claim
 7. 52. The method of claim 51, wherein the mammal is a human.53. A method for the treatment or amelioration of a disease resultingfrom decreased cell death or increased cell proliferation comprisingadministering to a mammal in need thereof a pharmaceutically effectiveamount of an antibody to a protein according to claim
 7. 54. The methodof claim 53, wherein the mammal is a human.
 55. A method for thetreatment or amelioration of a disease resulting from abnormal migrationor motility of cells selected from the group consisting of cancer,restenosis and diabetic neuropathy, the method comprising administeringto a mammal in need thereof, a pharmaceutically effective amount of anantibody to a protein according to claim
 7. 56. The method of claim 55,wherein the mammal is a human.
 57. An antibody specific for a proteinaccording to claim
 7. 58. The antibody according to claim 57, whereinthe antibody is a polyclonal antibody.
 59. The antibody according toclaim 57, wherein the antibody is a monoclonal antibody.